Main  Lib. 


BlOtOGY 

UBRAfW 

G 


A 


LABORATORY  GUIDE 


IN 


ELEMENTARY    BACTERIOLOGY 


BY 


WILLIAM  DODGE  FROST 

INSTRUCTOR  IN  BACTERIOLOGY,  UNIVERSITY  OF  WISCONSIN 


ILLUSTRATED 


1901 

PUBLISHED    BY    THE   AUTHOR 
MADISON,  WIS. 


THE  LIBRARY  OF 

CONGRESS, 
Two  COfifs  RECEIVED 

MAR,  20    1901 

COPYWOMT    ENTRY 


, 
SLABS  O.  XXc.  No. 

Afv?o 

COPY  A. 


F.S 


Main  Lib. 
Agria.  Uept, 


COPYRIGHT,  1901,  BY 
WILLIAM  DODGE  FROST. 


OUPLI1..1 


TRACY,    GIBBS   &   CO.,    PRINTERS, 
MADISON,  WIS. 


55 
S 


PREFACE. 


The.  following  pages  constitute,  substantially,  the  material  which  has  been  furnished  the  stu- 
dents in  Bacteriology  at  the  University  of  Wisconsin,  in  mimeograph  form,  for  several  years.  They 
contain  directions  for  the  performance  of  certain  fundamental  exercises  in  Bacteriology. 

In  a  rapidly  developing  subject  it  is  important  that  the  various  exercises  be  worded  so  as  to  lend 
themselves  readily  to  changes  which  become  desirable  from  time  to  time.  With  this  end  in  view  the 
exercises  have  been  divided,  where  possible,  into  a  general  and  a  special  part.  The  general  directions 
contain  the  essential  part  of  the  exercise  which  does  not  permit  of  any  considerable  variation,  while 
the  special  directions  embrace  such  features  as  are  most  subject  to  modification,  as  for  instance, 
the  particular  organism  to  be  used,  the  kind  of  medium,  the  incubation  temperature,  etc.  Desirable 
changes  here  are  easily  indicated  when  the  exercise  is  assigned. 

Some  of  the  exercises  can  be  performed  in  a  few  minutes,  while  others  require  several  days 
for  their  completion.  No  attempt  has  been  made  to  group  them  according  to  their  length,  nor  to 
divide  the  text  into  lessons,  but  as  far  as  possible  they  are  arranged  in  the  order  in  which  they  would 
be  logically  used  in  the  laboratory. 

The  right  hand  pages  have  been  left  for  notes  and  drawings  with  the  idea  that  notes  in  perma- 
nent form  are  the  only  ones  of  value  to  the  student  in  subsequent  years. 

The  charts  of  the  various  organisms  furnish  a  most  satisfactory  means  for  recording  the  observa- 
tions made  during  the  study  of  a  germ  and  are  especially  convenient  for  reference. 

Part  I.  is  the  work  required  of  students  taking  the  General  Course  in  which  special  emphasis  is 
placed  on  the  biology  of  bacteria.  It  is  completed  in  the  first  semester.  Part  II.  which  is  given 
during  the  second  semester  includes  the  more  specialized  phases  of  the  work,  particularly  as  applied 
to  the  student  preparing  for  medicine. 

References  have  been  made  to  all  of  the  leading  English  text-books  and  occasionally  to  original 
sources.  It  is  expected  that  the  student  will  make  constant  use  of  these  references. 

My  thanks  are  due  Prof.  H.  L.  Russell  under  whose  general  direction  the  work  outlined  here  is 
given,  for  valuable  help  in  the  selection  and  arrangement  of  the  material  and  for  generous  council.  I 
am  also  indebted  to  Mr.  E.  G.  Hastings,  Assistant  Bacteriologist  to  the  Wisconsin  Experiment  Station, 
for  critical  reading  of  manuscript  and  proof. 

WILLIAM  DODGE  FROST. 

MADISON,  Wis.,  January,  1901. 

(iii) 


263429 


CONTENTS. 


List  of  References vi 

List  of  Apparatus vii 

Laboratory  Rules viii 


PART  I.— GENERAL  BACTERIOLOGY. 
CHAPTER  I.     MORPHOLOGY  AND  ELEMENTARY  TECHNIQUE. 


EXERCISE.  PAGE. 

I.    Cleaning  Glassware 2 

II.    Plugging  Flasks  and  Tube? 2 

III.  Sterilization  of  Glassware 2 

IV.  Preparation  of  Bouillon   4 

V.  Filling  Test-tubes  and  Flasks  with 

Culture  Media 6 

VI.    Sterilization  of  Culture  Media 8 

VII.    Preparation  of  Gelatin 10 

VIII.    Preparation  of  Agar 10 

IX     Preparation  of  Potatoes 12 

X.    Preparation  of  Water-blanks 12 

XI.    Care  of  Culture  Media 14 

XII.    Platinum  Needles  14 

XIII     Test-tube  Cultures 14 

XIV.    Incubation  of  Cultures 16 

XV.  Cleaning  Slides  and  Cover-glasses.  18 

XVI.  Preparation  of  Staining  Solutions.  18 

XVII.  Simple  Cover-glass  Preparation. ..  20 

XVIII.    Use  of  Microscope    22 

XIX.    Hanging-drop  Preparation 24 


EXERCISE.  PAGE. 

XX.    Test-tube     Cultures      Illustrating 

Form  Types 26 

XXI.    Study  of  Test-tube  Cultures 26 

XXII.    Microscopical     Study     of     Form 

Types 26 

XXIII.    Drawing  Bacteria 28 

XXIV     Study  of  Cell  Grouping 30 

XXV.    Study  of  Involution  Forms 30 

XXVI.    Gelatin  Plate  Cultures 32 

XXVII.    Agar  Plate  Cultures 34 

XXVIII.    Roll  Cultures 34 

XXIX.    Study  of  Plate  Cultures 36 

XXX.    Use  of  Decolorizing  Agents 36 

XXXI.    Gram's  Stain 36 

XXXII.    Tubercle  Stain  (Gabbett) 38 

XXXIII.  Staining  Endospores 38 

XXXIV.  Study  of  Endospores 40 

XXXV.    Flagella  Stain 40 

XXXVI.    Capsule  Stain 42 


CHAPTER  II.     PHYSIOLOGY  OF  BACTERIA. 


EXERCISE.  PAGE. 

XXXVII     Preparation  of  Special  Media 44 

XXXVIII.    Effect  of    Reaction   of    Media  on 

Growth 44 

XXXIX.    Effect  of  Concentration  of  Media 

on  Growth   46 

XL.    Effect  of   Temperature   Variations 

on  Rate  of  Growth 46 

XLI.    Determination   of   Thermal   Death 

Point 46 

XLII.  Comparative  Efficiency  of  Dry  and 

Moist  Heat 48 

XLIII.    Effect  of  Desiccation 48 

XLIV.    Effect  of  Chemicals  on  Bacteria  .  .  48 

XLV.    Relation  to  Oxygen 50 

XLVI.    Effect  of  Direct  Sunlight 50 


EXERCISE.  PAGE. 

XLVII.  Detection  of  Gas 50 

XLVIII.  Quantitative  Analysis  of  Gas 50 

XLIX.  Detection  of  Acids 52 

L.  Quantitative  Determination  of  Acids  52 

LI.  Detection  of  Nitrites  in  Cultures. .  52 

LU.  Detection  of  Ammonia 52 

LI  1 1.  Detection     of     Sulphuretted      Hy- 
drogen    54 

LI V.  Detection  of  Indol 54 

LV.  Determination    of     Chemical    En- 
zymes in  Cultures 54 

LVI.  Variation  in  Enzyme  Production. .  54 

LVII.  Variation  in  Color  Production  ....  56 


CHAPTER  III.     TAXONOMY. 

PAGE.     I 

Points  to  be  observed  in  the  study  of  Bacteria. .     57   I   Classification  of  Bacteria  (Migula). 


PAGE. 
60 


CHAPTER  IV.     SYSTEMATIC  STUDY  OF  REPRESENTATIVE  NON-PATHOGENIC  BACTERIA. 


EXERCISE.  PAGE. 

LVIII.    Preparation  of  Special  Media 63 

LIX.    Bacillus  prodigiosus 64 

LX.    Variety  of  Pigments   66 

LXI.    Separation    of    Bacterial  Coloring 

Matter 67 


EXERCISE.  PAGE. 

LXII.    Bacterium  phosphorescens 68 

LXIII.    Bacillus  acidi  lactici 70 

LXIV.    Bacillus  vulgaris    72 


(iv) 


CHAPTER  V.     BACTERIOLOGICAL  ANALYSIS. 


EXERCISE.  PAGE. 

LXV.    Comparative  Analysis  of  Air 80 

LXVT.    Quantitative      Determination       of 

Number  of  Bacteria  in  Air 80 

LXVII.    Relation  of  Bacteria  in  Air  to  Dust 

Particles 82 

LXVIII.     Estimation  of  Number  of  Bacteria 

in  Soil . .  82 


EXERCISE.  PAGE. 

LXIX.    Water  Analysis 82 

LXX.    Quantitative  Analysis  of  Milk.    ...  84 

LXXI.    Efficiency  of  Pasteurization 84 

LXXII.    Testing  Antiseptic  Action  of  Chem- 
icals    84 

LXXIII.    Testing     Disinfecting     Action     of 

Chemicals 86 


PART  II.      MEDICAL  BACTERIOLOGY. 


CHAPTER  VI.     PATHOGENIC  AEROBES. 


EXERCISE.  PAGE. 

LXXIV.  Preparation  of  Culture  Media 88 

LXXV.  Streptococcus  pyogenes 90 

LXXV1.  Micrococcus  pyogenes *92 

LXXVII.  Micrococcus  melitensis 94 

LXXVIII.  Micrococcus  aureus 96 

LXX IX.  Micrococcus  gonorrhoeae 98 

LXXX.  Micrococcus  intracellularis  100 

LXXXI.  Sarcina  tetragena 102 

LXXXII.  Bacterium  anthracis 104 

LXXXIII.  Bacterium  pneumoniae 106 

LXXXI V.  Bacterium  pneumonicum 108 

LXXXV.  Bacterium  cuniculicida no 

LXXXVI.  Bacterium  rhusiopathiae 112 


EXERCISE.  PAGE. 

LXXX VII.  Bacterium  tuberculosis 114 

LXXXVIII.  Bacterium  mallei 116 

LXXXIX.  Bacterium  diphtheriae 1 18 

XC.  Bacterium  influenzae 120 

XCI.  Bacillus  typhosus 122 

XCII.  Bacillus  pestis     124 

XCIII.  Bacillus  suipestifer 126 

XCIV.  Bacillus  icteroides 128 

XCV.  Pseudomonas  aeruginosa 130 

XCVI  Microspira  comma 132 

XCVII.  Microspira  metschnikovi 134 

XCVIII.  Microspira  finkleri 136 


CHAPTER  VII.     PATHOGENIC  ANAEROBES. 


EXERCISE. 


XCIX.    Bacterium   welchii 150 

C.    Bacillus  chauvaei 153 


EXERCISE. 


CI.    Bacillus  oedematis  ....    154 

CII.    Bacillus  tetani 156 


CHAPTER  VIII.     ANIMAL  INOCULATION  AND  STAINING  OF  BACTERIA  IN  TISSUE. 


EXERCISE.  PAGE. 

CIII.    Animal  Inoculation 162 


EXERCISE.  PAGE. 

CIV.    Preparation  of  Tissue    for  Exami- 
nation    170 

CV.    Staining  Sections IJ2 


CHAPTER  IX.     BACTERIOLOGICAL  DIAGNOSIS. 


EXERCISE.  PAGE. 

CVI.  Examination  of  Buccal  Secretion. .    178 

CVII.    Examination  of  Sputum 180 

CVIII.    Examination  of  Blood 184 

CIX.    Examination  of  Faeces 188 

CX.    Examination  of  Urine 192 


EXERCISE.  PAGE. 

CXI.    Examination   of   Transudates   and 

Exudates 194 

CXII.    Diagnosis  of  Rabies 198 

CXIII.    Examination  of  Material  from  Hu- 
man Autopsies 198 


CHAPTER  X.     DETECTION  OF  PATHOGENIC  BACTERIA  IN  WATER  AND  MILK  SUPPLIES. 


EXERCISE.  PAGE. 

CXIV.    Examination  of  Water   for   Patho- 
genic Bacteria 200 


EXERCISE.  PAGE. 

CXV.   Examination   of   Milk  for    Patho- 
genic Bacteria 200 


[v] 


LIST  OF  TEXTS  AND  REFERENCE  WORKS  WITH  ABBREVIA- 
TIONS USED. 


A.—  Abbott:  Principles  of  Bicteriology.     Lea  Bros.  '&  Co.    Philadelphia,  5th  Edit..  1899. 

B. —  Bowhill:  Manual  of  Bacteriological  Technique.      Oliver  &  Boyd,  London.  1899. 

F. —  Fischer:  Structure  and  Functions  of  Bacteria.     Clarendon  Press,  New  York,  1900. 

Fr. —  Frankland:   Micro-organisms  of  Water.      Longmans,  Green  &  Co  ,  1894. 

G.   -  Gage:  The  Microscope.     Comstock  Pub.  Co.,  Ithaca,  N.  Y.,  7th  Edit.,  1899. 

H.—  Hewlett:  Manual  of  Bacteriology.      Blakiston,  Son  &  Co.,  Philadelphia,  1898. 

J.  H. —        Jordan's  Translation  of  Hueppe:   Principles  of    Bacteriology.     Opan    Court  Pub.    Co.,    Chicago, 

1899. 

v.  J. —        v.  Jaksch:  Clinical  Diagnosis.     Charles  Griffin  &  Co.,  London,  4th  Edit.,  1899. 
K.  &D.—  Kanthack  &  Drysdale:  Practical  Bacteriology.     MacMillan  Co.,  New  York,  1895. 
L. —  Lafar:  Technical  Mycology.     Vol.  i.     Lippencott  Co  ,  Philadelphia,  1898. 

L.&  K. —  Levy  &  Klemperer:  Clinical  Bacteriology.     Saunders  &  Co.,  Philadelphia,  1900. 
L.  &  N. —  Lehmann  &  Neumann:   Atlas  and  Essentials  of  Bacteriology.     Wood  &  Co.,  New  York,  1897. 
M. —  Moore:  Laboratory  Directions  for  Beginners  in  Bacteriology.     Ginn  &  Co  ,  New  York,  1000. 

M.  &  R. —  Muir  &  Ritchie:  Manual  of  Bacteriology.     MacMillan  Co  ,  New  York,  2nd  Edit.,  1899. 
M.  &  W.— Mallory  &  Wright:  Pathological  Technique.     Saunders  &  Co  ,  Philadelphia,  1897. 
McF. —       McFarland:  Text-Book  of  Pathogenic  Bacteria.    Saunders  &  Co.,  Philadelphia,  2nd  Edit.,    1898. 
N. —  Novy:  Laboratory  Work  in  Bacteriology.     Geo.  Wahr,  Ann  Arbor,  Mich.,  2nd  Edit.,  1899 

Ne. —  Newman:  Bacteria.     Putnam,  New  York,  1899. 

P. —  Park:  Bacteriology  in  Medicine  and  Surgery.     Lea  Bros.  &  Co. ,  Philadelphia,   1899. 

P.  B.  C  —  Proceedings  of  the  Bacteriological  Committee  from   Jour.  Amer.     Pub.  Health  Assn.     Vol.  XXII. 
P.  &  M. —   Peamain  &  Moor:  Applied  Bacteriology.     Bailliere,  Tindall  &  Cox,  London,  1897. 
S. —  Sternberg:   Manual  of  Bacteriology.     Wood  &  Co. ,  New  York,  1893. 

Si. —  Simon:  Clinical  Diagnosis.     Lea  Bros.  &  Co.,  Philadelphia,  2d  Edit.,  1897. 

W.—  Woodhead:  Bacteria  and  Their  Products.     Charles  Scribner  &  Sons,  New  York,  1892. 

Wm. —        Williams:  Manual  of  Bacteriology.     Blakiston,  Son  &  Co.,  Philadelphia,  1898. 

[vi] 


LIST  OF  APPARATUS. 


This  list  comprises  the  apparatus  which  is  to  be  under  the  exclusive  control  of  the  student  and 
does  not  include  the  general  laboratory  outfit,  such  as  sterilizers,  incubators,  microscopes,  general 
chemical  supplies,  etc. 


FOR  INDIVIDUAL  USE. 

A. 

50  (|  oz.)  cover-glasses,  18mm.  (Jin.)  square 

and  0.17  mm.  thick  (No.  2). 
50  glass  slides. 
100  labels,  2  cm.  square. 
13  cm.  platinum  wire  (No.  27). 
1  pair  cover-glass  forceps  (Cornet  or  Stew- 
art). 

1  pair  fine  pointed  forceps. 

2  slide  boxes  for  50  slfdes. 
1  hanging-drop  slide. 

1  towel. 

B. 

1  flask,  lOOOcc. 
1  flask,    400  cc. 

3  flasks,  250  cc. 

1  flask,    100  cc. 

200  test-tubes  (15  X  120  mm.). 
15  Petri  dishes  (10  cm). 

2  fermentation  tubes. 
2  glass  tumblers. 

4  tin  cans. 

2  glass  rods  for  platinum  needles. 

3  pipettes,  1  cc. 

1  brass  tube  to  hold  pipettes  (25  X  250  mm.). 

8  stain  bottles  with  pipettes,  in  block. 

1  waste  dish. 

1  yard  of  muslin. 

3  sheets  of  filter  paper. 

3  sheets  of  lens  paper. 


FOR  GROUP  USE  (About  Four  Students). 

1  glass  funnel,  12  cm. 
1  glass  funnel,  5  cm. 

1  filtering  flask  with  rubber  stopper. 

2  stirring  rods. 
1  pipette,  5  cc. 

1  thermometer,  0-100"  C. 
10cm.  rubber  tubing.  1  cm.  dia      See  Fig.  1. 
1  Mohr  stopcock. 
1  anaerobic  jar  for  plates. 
1  anaerobic  jar  for  tubes. 
1  potato  knife. 

1  Bunsen  burner  witli  tubing. 
1  piece  of  wire  gauze. 
1  tripod  with  reducing  rings 
1  rice  cooker. 

3  small  wire  baskets. 
1  enamel  pan. 

1  roll  of  cotton  wool. 
YT,  lb.  absorbent  cotton. 
1  piece  of  Russia  iron,  12  cm.  square. 
1  graduated  cylinder,  300  cc. 
1  graduated  cylinder,  100  cc. 
1  graduated  cylinder,  25  cc. 
1  evaporating  dish,  10  cm. 
1  disinfecting  jar. 
1  copper  cup. 
1  ring  stand  with  clamp. 
1  test-tube  brush. 


[vii] 


LABORATORY  RULES. 


I.  Food  should  not  be  eaten  in  the  laboratory  and  lead  pencils  or  labels  should 
not  be  moistened  with  the  tongue. 

II.  All  possible  cleanliness  should  be  observed  in  the  care  of  apparatus,  desk, 
etc. 

III.  The  platinum  needles  used  in  making-  cultures  should  be  sterilized  shortly 
before  and  immediately  after  use  and  before  they  are  laid  down.    When  the 
needles  are  covered  with  infectious  material  they  should  be  held  at  the  side  of 
the  flame  until  dry  before  being-  sterilized;  this  will  avoid  the  danger  of  scatter- 
ing this  material  about  the  laboratory. 

IV.  If  infectious  matter  should  by  accident  come  in  contact  with  the  hands  or 
be  dropped  on  the  table  or  floor,  corrosive  sublimate  (1:1000)  should  be  imme- 
diately applied. 

V.  Solid  material,  culture  media  and  corrosive  sublimate  should  not  be  put 
in  the   sink  but  in  crocks  provided  for  the  purpose.    Burnt  matches,  pieces  of 
paper,  etc.,  should  also  be  put  in  the  crocks  and  not  on  the  floor. 

VI.  All  cultures  of  bacteria  should  be  labeled  with  the  name  of  the  organism, 
the  name  of  the  student  and  the  date. 

VII.  Discarded  cultures  shouldbe  covered  with  corrosive  sublimate  and  placed 
in  a  proper  receptacle  and  under  no  condition  should  they  be  left  lying  about 
the  laboratory.    Pipettes  which  have  been  used  to  handle  infectious  material 
should  be  placed  in  a  glass  cylinder  containing  a  disinfectant  or  potassium  bi- 
chromate and  sulphuric  acid. 

VIII.  Whan  using  the  steam  sterilizer  see  that  there  is  enough  water  present 
before  lighting  the  gas  and  do  not  leave  the  laboratory  until  the  gas  has  been 
turned  off. 

IX.  Before  beginning  an  exercise  read  over  the  directions  and  look  up  some 
of  the  references.    Keep  notes  of  everything  done  and  the  conclusions  reached 
on  the  right  hand  pages  in  this  Guide.    Make  drawings  wherever  they  will  be 
of  value.    Outline  with  pencil  and  fill  in  with  India  ink.     The  laboratory  Guide 
should  be  kept  in  the  laboratory. 

X.  At  the  close  of  the  day's  work  the  tables  should  be  washed  with  corrosive 
sublimate  and  the  hands  disinfected  by  washing  in  the  sublimate  solution  (or  a 
germicidal  soap)  and  then  in  soap  and  water. 

[viii] 


PART    I. 

GENERAL  BACTERIOLOGY 


PART  I.— GENERAL  BACTERIOLOGY. 
CHAPTER  I. 

MORPHOLOGY  AND  ELEMENTARY  TECHNIQUE. 


EXERCISE  I.  CLEANING  GLASSWARE. 

GENERAL  DIRECTIONS.  All  glassware  to  contain  culture  media  must  be  thoroughly 
clean.  New  glassware  should  be  washed  in  hot  soap-suds  (a  test-tube  brush  will  be 
needed  for  the  test-tubes) ,  rinsed  in  tap  water  and  then  placed  for  a  few  minutes  in  water 
to  which  about  1%  of  hydrochloric  acid  has  been  added  to  remove  free  alkali  frequently 
present  on  new  glass,  and  then  thoroughly  rinsed  in  tap  water.  It  is  then  allowed  to 
drain.  Test-tubes  and  flasks  are  best  dried  by  placing  them  on  a  drain  board  especially 
prepared,  or  standing  them  mouth  down  in  a  box  with  a  cloth  bottom  or  on  filter  paper. 

Glassware  containing  media  (discarded  cultures,  etc.),  is  best  cleaned  by  first  stand- 
ing in  water  for  some  hours,  or  by  being  steamed  and  pouring  out  the  material  while  in 
a  liquid  condition  and  then  cleaning  as  above  with  the  exception  of  the  use  of  the  hydro- 
chloric acid. 

REFERENCES.     A.    120;  H.  39;  K.  &  D.  81;  M.  &  W.  74;  N.  158;  P.  223. 

SPECIAL  DIRECTIONS.  Clean  as  directed  above,  all  flasks,  test-tubes,  fermentation 
tubes  and  Petri  dishes  in  your  possession. 

EXERCISE  II.  PLUGGING  FLASKS  AND  TUBES. 

GENERAL  DIRECTIONS.  When  the  flasks,  test-tubes  and  fermentation  tubes  are 
thoroughly  dry  they  are  to  be  plugged  with  cotton.  The  cotton  for  this  purpose  should 
be  of  the  best  non-absorbent  quality,  i.  e.,  as  free  from  foreign  matter  as  possible.  The 
plugs  should  be  sufficiently  loose  to  permit  the  interchange  of  gases  and  at  the  same  time 
tight  enough  to  support  the  weight  of  the  vessel  and  its  contents,  to  prevent  their  being 
pulled  out  in  handling  the  vessel.  The  cotton  should  be  rolled  into  a  cylinder  of  the 
proper  diameter  and  long  enough  to  extend  into  the  mouth  about  2?  cm.  (1  in.)  and  pro- 
ject sufficiently  to  protect  the  lips  from  dust.  The  plug  should  be  pushed  in  straight  and 
not  twisted;  the  surface  next  to  the  glass  must  be  perfectly  smooth,  presenting  no 
creases  for  the  entrance  of  dust. 

REFERENCES.     A.  121;  H.  39;  M.   &  W.  74;  M.  &  R.  56;  McF.107;  P.  223. 
SPECIAL  DIRECTIONS.     Plug   all  test-tubes,  flasks  and  fermentation  tubes  in  your 
possession. 

EXERCISE  III.     STERILIZATION  OF  GLASSWARE. 

GENERAL  DIRECTIONS.  The  glassware  thus  prepared  is  ready  for  sterilization,  which 
process  is  accomplished  in  an  apparatus  called  the  hot  air  sterilizer.  This  is  a  sheet  iron 
or  copper  box  with  a  double  wall  which  permits  of  rapid  heating.  The  apparatus  should 


General  Bacteriology. 


be  so  arranged  that  a  temperature  of  150°  C.  can  be  quickly  reached  and  readily  main- 
tained. In  such  a  sterilizer  all  glassware  to  be  used  for  the  reception  of  culture  media, 
such  as  flasks,  test-tubes,  Petri  dishes,  etc.,  is  submitted  to  a  temperature  of  140-150°  C. 
for  1  hour,  or  until  the  cotton  plugs  are  slightly  browned;  this  change  being  due  to  the 
incipient  charring  of  the  cotton.  The  test-tubes  are  placed  erect  in  square  baskets  made 
of  galvanized  iron  wire.  When  the  air  in  the  sterilizer  has  cooled  to  about  40°  C.  the 
glassware  can  be  taken  out  and  stored  ready  for  use.  The  Petri  dishes  are  not  to  be  opened 
until  used  for  culture  purposes. 

REFERENCES.  A.  71  and  121;  H.  32;  L.  &  K.  74;  M.  &  R.  36;  N.  159;  McF. 
106;  P.  223;  S.  51. 

SPECIAL  DIRECTIONS.  All  glassware  prepared  in  I.  is  to  be  sterilized  for  one 
hour  at  150°  C.  The  small  pipettes  should  be  placed  in  brass  tubes,  provided  for  the 
purpose,  and  also  sterilized. 

EXERCISE  IV.     PREPARATION  OF  BOUILLON. 

GENERAL  DIRECTIONS.  Any  one  of  the  three  methods  (A  B  or  C)  may  be  used. 
They  are  arranged  in  order  of  preference,  but  method  C  is  the  most  convenient,  and 
hence  most  used. 


A. 


B. 

Secure  meat  as 
under  A  a,  add  1  liter 
of  distilled  water, 
weigh  (see  e  below), 
cook  for  ^  hour  at 
about  70°  C. ,  and  pro- 
ceed as  directed  under 
e  below. 


Weigh  out  three 
grams  of  beef  extract 
(such  as  Liebig's), 
add  1  liter  of  water, 
and  then  proceed  as 
directed  under  e  be- 
low. 


a.  From  500  grams  (1^-lb.)  of  lean 
beef,  remove  the  fat  and  connective  tis- 
sue and  mince  (Hamburg  steak). 

6 .  Add  1  liter  of  distilled  water  and 
after  thoroughly  shaking  set  in  ice  chest 
for  12  to  24  hours. 

c.  Squeeze  through  a  cloth  and  add 
enough  distilled  water  to  make  1   liter 
and  place  in  vessel  to  cook.  This  may  be 
done  either  in  a  flask  which  is  heated  in  a 
water-bath  or  a  sterilizer,  or  in  a  rice 
cooker.  In  this  case  use  a  50  %  solution  of 
calcium  chloride  in  outer  vessel  instead 
of  water  as  by  this  means  the  contents 
of  the  inner  vessel  can  be  brought  to  a 
rapid  ebullition,  something  impossible  by 
the  use  of  water  alone. 

d.  Boil  \  hour  and  make  up  loss  of 
water. 

e.  Add  to  any  of  the  above  solutions: 

1%  (10  gms.)  peptone  (Witte)  and -5-%  (5  gms.)  common  salt  (NaCl),  then  weigh 
solution,  with  vessel,  so  that  the  water  which  is  subsequently  driven  off  in  cooking  can 
be  accurately  replaced. 

/.  Heat  until  ingredients  are  in  solution,  then  restore  the  water  lost  by  evaporation. 

g.  Neutralize  or  render  slightly  alkaline.  This  is  a  very  important  step  and  calls  for 
great  care.  Method  A  is  more  accurate  and  should  be  employed  for  special  or  research 
work.  For  ordinary  routine  work  B  may  be  employed. 


C. 


General  Bacteriology. 


B. 

Use  a  normal  solution  of  so- 
dium hydroxide  (yNaOH).  Add 
to  the  hot  solution  a  few  cc.  at  a 
time,  at  first,  later  a  few  drops, 
stirring  thoroughly  with  a  glass 
rod.  After  each  addition,  test 
by  placing  a  drop  of  the  solu- 
tion by  means  of  the  glass  rod 
on  a  strip  of  red  litmus  paper, 
and  then  moisten  the  paper  with 
distilled  water.  The  addition 
should  continue  until  the  red 
litmus  paper  is  turned  blue,  but 
no  change  occurs  on  blue  litmus 
paper. 


A. 

1 . )  Titrate  as  follows :  Pipette  off  5  cc:  of  the  fluid 
into  a  4-inch  evaporating  dish,  add  45  cc.  of  distilled 
water,  boil  for  three  minutes,  add  1  cc.  of  phenol- 
phthalein  (0.5%  substance  in  50%  alcohol),  and  then 
carefully  run  in,  drop  by  drop,  from  a  burette  a  twen- 
tieth normal  *  solution  of  sodium  hydroxide  (^VNa 
OH)  until  the  solution  turns  a  faint  pink  color.  Treat 
two  other  samples  in  the  same  way.  If  the  amount 
of  Na  OH  required  is  approximately  the  same  in  each 
case  the  average  can  be  taken  as  the  amount  necessary 
to  neutralize  5  cc.  Calculate  the  amount  necessary 
to  neutralize  the  whole  (1000-15  cc.).  Since  this 
amount  would  dilute  the  medium  too  much,  a  stronger 
solution  (normal)  is  used,  hence, 

2.)  Neutralize  by  adding  ^Vth  of  the  volume  cal- 
culated above  of  a  normal  solution  of  sodium  hydrox- 
ide. Test  the  accuracy  of  the  work  at  this  point  by 
the  addition  of  a  few  drops  of  phenolphthalein  to  a 
cc.  or  so  of  the  medium.  If  a  faint  pinkish  tint  is  not 
obtained,  titration  and  neutralization  must  be  re- 
peated. 

If  by  mistake  more  alkali  is  added  than  is  required,  the  reaction  can  be  corrected  by 
the  use  of  a  normal  solution  of  hydrochloric  acid. 

h.  Boil  for  5  minutes  and  restore  weight. 

i.  Test  reaction  and  adjust  if  necessary. 

j.  Add  0.5  to  1.5%  of  a  normal  hydrochloric  acid  if  neutralized  by  method  A,  oth- 
erwise omit.  The  amount  of  acid  to  be  added  varies  with  the  purpose  for  which  the 
medium  is  to  be  used,  e.  g.,  in  water  analyses  +1.5  (acid)  is  preferable,  with  the  path- 
ogenic bacteria  a  smaller  amount  of  acid  (+  0.5)  more  nearly  meets  requirements. 

k.  Filter  through  moistened  filter  paper  (Abbott  p.  96),  or  absorbent  cotton, 
(VII.  m).  If  the  filtrate  is  not  perfectly  clear,  cool  to  60°  C.,  add  the  white  of  an 
egg,  thoroughly  mix  and  boil  for  5  minutes  without  stirring. 

The  filtrate  (bouillon)  should  be  of  a  light  straw  color,  perfectly  clear,  and  should 
not  give  a  precipitate  on  boiling. 

REFERENCES.     A.  90;  M.  &  R.  43;   McF.  124;  N.  234;    P.  212;   P.   B.  C.  18-24. 

SPECIAL  DIRECTIONS.  Prepare  1  liter  of  bouillon  according  to  method  C. 
Secure  and  put  to  soak  meat  for  VII. 

EXERCISE  V.     FILLING  TEST-TUBES  AND  FLASKS  WITH  CULTURE  MEDIA. 

GENERAL  DIRECTIONS.  In  filling  tubes  be  careful  not  to  allow  the  media  to  touch 
the  neck  of  the  vessels  as  this  will  cause  the  cotton  to  stick  to  the  glass  when  the  plugs 
are  removed.  Place  the  culture  fluid  to  be  tubed  in  a  funnel  arranged  with  a  delivery 


•Normal  solutions  are  prepared  so  that  one  liter  at  16°  C.  shall  contain  the  hydrogen  equivalent 
of  the  active  reagent  weighed  in  grams  (Sutton).  For  present  purposes  a  4  %  solution  of  sodium 
hydrate  is  sufficiently  accurate. 


8 


General  Bacteriology. 


tube  and  stopcock  (fig.  1),  from  which  it  can  be  run  into  sterile  vessels.     Test-tubes 
should  contain  6-10  cc.  of  medium  (about  3  cm.  deep).     Flasks  are  to  be  filled  about 

three-fourths  full.  

SPECIAL  DIRECTIONS.      Fill  15  test-tubes  .  and  preserve   remainder 
of  bouillon  in  larger  flasks. 


EXERCISE  VI.     STERILIZATION  OF  CULTURE  MEDIA. 


O    f 


FIG.  1.  Appara- 
tus for  filling  test- 
tubes. 


EXPLANATORY.  To  accomplish  this  steam  is  used  almost  exclusively 
either  as  streaming  steam  or  under  pressure.  The  unconfined  steam  is 
applied  in  an  apparatus  known  as  a  steam  sterilizer.  Of  the  various 
patterns  the  Arnold  is  perhaps  the  most  satisfactory.  It  is  effective, 
economical  in  the  use  of  gas,  and  does  not  allow  the  escape  of  large 
quantities  of  steam  into  the  room,  as  a  large  part  is  condensed  to  be  re- 
converted into  steam.  For  student  use  the  form  shown  in  fig.  2  is  very 
convenient.  The  method  of  using  these  different  forms  is  identical. 
Always  have  plenty  of  water  present  before  heating.  The  discontinuous  method  is  most 
frequently  employed.  Exposure  is  made  on  three  consecutive  days  for  20  minutes,  be- 
ginning to  count  time  when  the  material  reaches  the  temperature  of  the  steam,  which  will 
vary  with  different  substances  and  the  volume  treated.  Between  successive  steamings 
culture  media  should  be  kept  under  conditions  favorable  to  bacterial  development  (room 
or  incubator  temperature). 

For  the  employment  of  steam  under  pressure  the 
autoclave  is  essential.  The  lid  should  contain  a  thermom- 
eter as  well  as  a  steam  gauge,  safety  and  outlet  valve.  A 
thermo-regulator  is  also  desirable.  The  following  table 
gives  the  temperature  corresponding  to  atmospheres  of 

pressure : 

Atmospheres.  Degrees  C. 

1  100 
1.5  112.2 

2  121.4 

2.5  128-.8 

3  135.1 

This  table  is  only  true  when  all  of  the  air  in  the  apparatus  is  replaced  by  steam, 
and  hence  the  steam  must  be  allowed  to  escape  freely  before  the  outlet  valve  is  closed.  A  sin- 
gle exposure  of  20  minutes  at  a  temperature  of  120°  C.  (one  additional  atmosphere)  is 
sufficient  to  kill  all. germ  life.  After  the  proper  exposure,  care  must  be  taken  not  to  allow 
the  steam  to  escape  too  rapidly,  otherwise  the  culture  media  may  be  forced  against  the 
plugs  owing  to  the  unequal  pressure. 

GENERAL  DIRECTIONS.  Ordinary  media  may  be  sterilized  by  either  method.  Sugar 
media  cannot  be  sterilized  in  the  autoclave  as  it  must  not  be  heated  above  100°  C.  The 
solidifying  property  of  gelatin  is  impaired  if  submitted  to  a  temperature  of  120°  C. 
longer  than  15  minutes,  and  at  a  temperature  above  120°  C.  momentarily. 

REFERENCES.     A.  55-73;   M.  &  R.  37;   McF.  109;   N.  161;   P.  213. 

SPECIAL  DIRECTIONS.  Sterilize  bouillon  prepared  in  IV.  for  20  miuutes  in  a 
steam  sterilizer  oa  three  consecutive  days. 


FIG.  2.  Simple  sterilizer  consisting 
of  a  galvanized  iron  pail  with  a  cover 
a  and  a  false  bottom  b. 


10  General  Bacteriology. 

N.  B.  Some  time  is  required  to  raise  the  temperature  of  the  media  to  that  of  the  steam, 
especially  if  the  vessels  are  large. 

All  media  should  be  carefully  examined  every  day  for  a  week  or  more,  and  if  "specks" 
or  the  least  cloudiness  appears,  the  medium  is  not  sterile  and  the  process  of  sterilization 
must  be  repeated. 

All  receptacles  containing  media  should  be  labeled  after  sterilization.  For  this  pur- 
pose labels  can  be  purchased,  the  size  used  for  glass  slides,  or  gummed  paper  in  sheets 
can  be  cut  into  squares  (2  cm.).  The  labels  are  to  be  attached  to  each  vessel  1  cm.  from 
the  lip.  The  kind  of  medium  and  the  date  of  preparation  should  be  written  across  the 
top,  as  io"is-L>99  leaving  the  rest  of  the  label  to  be  filled  in  when  the  medium  is  inoc- 
ulated. 

EXERCISE  VII.     PREPARATION  OF  GELATIN. 

GENERAL  DIRECTIONS. 

a  to  d.  Same  as  bouillon.     (IV.) 

e.  Add  1%  peptone;  0.5%  salt  and  10-15%  *  of  best  white  gelatin,  and  weigh. 

/.  Heat  until  ingredients  are  dissolved. 

g.  Neutralize. 

h.  Boil  5  minutes  and  restore  weight. 

i.  Test  reaction. 

j.  If  neutralized  by  method  A  add  5  cc.  of  a  normal  hydro- 
chloric acid.  In  method  B  omit  acid. 

k.  Cool  and  add  egg  and  boil  5  minutes. 

I.  Filter.  Arrange  the  apparatus  shown  in  fig.  3.  Use 
absorbent  cotton.  The  funnel  and  flask  should  first  be  heated 
with  warm  water.  Start  the  filter  pump  before  pouring  in  the 
culture  medium.  This  prevents  the  unfiltered  gelatin  from  TiTsTT^aratus  for  filtering 

passing  between  the    COtton  and  the  glass.  media  through  absorbent  cotton; 

"'  layer  of  cotton:  *•  tubes  for 


Tnhp    (V  } 
"*•    J  making    connection    with     air 

W.    Sterilize.  pump;  ct  Bunsen  valve  to  prevent 

-r     •,     i  entrance  of  water  into  flasks. 

o.  Label. 

REFERENCES.     A.  95;   H.  42;  M.  &  R.  46;  McF.  127;  N.  153;  P.  B.  C.  26. 

SPECIAL  DIRECTIONS.  Make  1  liter,  using  method  A.  Fill  30  test-tubes.  Put 
the  remainder  in  flasks,  sterilize  in  steam  sterilizer  or  autoclave.  Remember  long  exposure 
to  high  heat  injures  the  solidifying  properties  of  gelatin. 

EXERCISE  VIII.    PREPARATION  OF  AQAR. 

GENERAL  DIRECTIONS. 

a.  Add  15  grams  of  agar-agar  threads  (finely  chopped)  to  500  cc.  of  water  and  either 
(l)boil  until  the  agar-agar  is  dissolved  (about^hour)  and  make  up  loss  of  water  by  evap- 
oration, or  (2)  dissolve  in  autoclave  by  heatingup  to  120°  C.,  closing  off  gas  and  allow- 
ing to  cool. 

*  The  amount  to  be  varied  according  to  the  season  of  the  year,  10  per  cent  in  winter,  12-15  per 
cent  in  summer,  but  it  should  be  remembered  that  different  quantities  affect  the  appearance  of  the 
culture. 


12 


General  Bacteriology. 


b.  l)-4)  Same  as  a-d  in  the  preparation  of  bouillon  (IV.),  except,  that  only  one- 
half  (500  cc.)  of  the  amount  of  water  is  added  to  the  beef  or  extract. 

5)  Add  1%  peptone  and  0.5%  salt. 

6)  Heat  until  peptone  is  dissolved. 

7)  Neutralize. 

8)  Mix   a.  and  b.  (in  case  beef  is  used  it  will  be  necessary  to  cool  a.  to  about  60° 
C.  before  mixing). 

9)  Boil  5  minutes  and  restore  weight. 

10)  Test  reaction. 

11)  Addition  of  egg  will  be  necessary  only  where  extract  is  used. 

12)  Filter  as  incase  of  gelatin,  (IV.  m.) 

13)  Tube. 

14)  Sterilize  in   steam   for  15   minutes  on  three  suc- 
cessive days  or   in  autoclave   for  20  minutes  at  120°  C. 
After  the  last  sterilization  place  most  of  the  tubes  in   a 
sloping  position  to  harden   (fig.  4),  these  are  known    as 

agar  slopes.     Those  solidified  horizontally  can  be  used  for  plate  cultures. 

15)  Label. 

REFERENCES.  A.  100;  H.  43;  M.  &  R.  48;  McF.  129;  N.  235;  P.  B.  C.  27;  S. 
43;  Journal  of  Applied  Microscopy,  1898,  1;  106. 

SPECIAL  DIRECTIONS.  Use  meat  extract,  make  1  liter,  fill  25  tubes  and  after  last 
sterilization  incline  20  of  them.  Place  the  remainder  in  flasks  and  sterilize. 


FIG.  4.    Method  of  sloping  agar. 


(23 


EXERCISE  IX.    PREPARATION  OF  POTATOES.     (BOLTON.) 

GENERAL  DIRECTIONS. 

a.  Select  a  number  of  rather  large  test-tubes   (150x20  mm.)  place  a 
small  wad  of  absorbent  cotton  in  the  bottom  of  each  (fig.  5  a),  plug  and 
sterilize  as  usual. 

b.  Wash  a  large  potato,  then  with  a  cork  borer  slightly  smaller  than 
the  test-tubes  punch  out  cylinders  about  5-6  cm.  long. 

c.  Divide  these  diagonally  and  trim  to  shape  indicated  in  fig.  5  b. 

d.  Add  a  few  drops  of  distilled  water  to  each  test-tube   and  place 
pieces  of  potato  in  position. 

e.  Sterilize  on  three  consecutive  days  for  30  to  45  minutes. 

Unless  the  tubes  are  to  be  used  immediately,  they  should  be  sealed. 
(XI.)  The  dark  color  can  be  prevented  by  immersing  the  pieces  between  c 
and  d  in  running  water  for  from  12-18  hours. 

REFERENCES.     A.  104;  M.  &  R.  54;  McF.  134;  N.  183;  P.  216;  P.  B.  C.  28;  S.  47. 

SPECIAL  DIRECTIONS.  Prepare  15  test-tubes  of  potato,  sterilize,  label,  and  seal  with 
paraffin.  (XI.  2.) 

EXERCISE  X.     PREPARATION  OF  WATER-BLANKS. 

GENERAL  DIRECTIONS.  Water-blanks  are  prepared  by  placing  exactly  10  cc.  of  a 
physiological  salt  solution  (6  gms.  per  1,000  cc.  of  water)  in  test-tubes  and  sterilizing  in 
autoclave  15  minutes  at  120  °  C.,  or  in  steamer  15  minutes  on  three  successive  days. 

SPECIAL  DIRECTIONS.     Prepare  and  sterilize  10  water-blanks. 


0. 

FIG.  5.  Bolton's 
potato  tube. 


14  General  Bacteriology. 

EXERCISE  XI.    CARE  OF  CULTURE  HEDIA. 

When  sterile  culture  media  (or  test-tube  cultures)  are  to  be  kept  for  some  time  they 
must  be  protected  from  evaporation  and  stored  in  a  dark,  cool  place.  Evaporation  may 
be  checked  to  a  considerable  extent,  (1)  by  storing  them  iu  tin  cans,  e.  g.  quinine  cans. 
Care  must  be  taken,  however,  that  these  do  not  become  too  damp,  in  which  case  the 
mould  fungi  frequently  grow  through  the  cotton  plugs;  (2)  flasks  and  test-tubes  may 
be  sealed  by  removing  the  plugs,  dipping  same  in  melted  paraffin  (melting  point  about 
50°  C.)  and  then  replacing  them;  (3)  by  cutting  off  the  projecting  cotton  and 
drawing  over  the  mouth  of  the  vessel  a  rubber  cap  (made  for  the  purpose)  which  has 
been  sterilized  in  a  solution  of  mercuric  bichloride  (1:  1,000,  spoken  of  in  the  lab- 
oratory as  "sublimate  solution") ;  or  (4)  By  use  of  a  cap  of  tin-foil.  In  this  case  the 
foil  should  be  put  on  as  soon  as  the  tubes  are  filled,  and  sterilized  with  the  medium. 

All  media  should  be  carefully  examined  every  day  for  a  week  or  more,  and  if  spots  or 
the  least  cloudiness  appears,  the  medium  is  not  sterile  and  the  process  of  sterilization  must 
be  repeated. 

EXERCISE  XII.     PLATINUM  NEEDLES. 


GENERAL  DIRECTIONS.     These  are  made  by  — C  \ 

fusing   a  piece  of  No.  27   platinum  wire   (5  cm. 
long)  into  a  glass  rod  or  tube  (18  cm.  long) .    (Fig. 

6.)  Each  student  should  have  two  such  needles;  FlG'8-  platinum  needles- 

in  one  the  wire  should  be  straight  (designated  "needle")  and  the  other  bent  to  form 
a  "loop".  This  loop  should  be  formed  around  a  No.  10  wire.  These  instruments  must 
be  sterilized  shortly  before  and  immediately  after  use  by  heating  the  wire  to  a  glow  in 
the  gas  flame.  The  handle  should  also  be  passed  through  the  flame  two  or  three  times. 
Cool  before  using.  If  the  habit  of  sterilizing  is  thoroughly  acquired  much  trouble  will 
be  avoided  and  possible  danger  prevented.  These  needles  will  be  in  constant  use. 

REFERENCES.     A.  125;  M.  &  B.  58;  N.  172;  P.  B.  C.  33,  foot  note. 

EXERCISE  XIII.    TEST-TUBE  CULTURES. 

EXPLANATORY.  The  extreme  minuteness  and  slight  variation  in  the  form  of  dif- 
ferent bacteria  render  a  thorough  study  of  them  by  direct  microscopic  observation  a  dif: 
ftcult  and  well  nigh  impossible  task.  In  their  study,  therefore,  it  is  necessary  to  depart 
from  the  usually  accepted  rules  that  govern  the  determination  of  the  life  history  of  other 
forms  of  life  and  resort  to  special  methods.  The  most  successful  of  these  are  those 
known  as  culture  methods.  According  to  these  methods  the  bacteria  are  sown  on  vari- 
ous food  substances  and  upon  these  they  develop  forming  masses  easily  visible  to  the 
naked  eye.  The  manner  of  their  growth  and  the  changes  which  they  produce  in  these 
media  make  it  possible  to  detect  differences  which  would  otherwise  escape  attention. 
The  most  common  culture  media,  bouillon,  gelatin,  agar  and  potato  have  already  been 
prepared,  and  others  will  be  described  as  needed. 

Cultures  maybe  made  either  in  test-tubes  (streak  or  stab  cultures),  or  on  glass 
plates,  as  plate  cultures.  The  plate  culture  is  especially  important  and  is  used  (a)  to 
obtain  pure  cultures;  and  (6)  for  ascertaining  the  character  of  the  colonies  as  an  aid  to 


16 


General  Bacteriolv;/ >/ . 


FIG.  7.     Method  of  holding  test-tubes. 


their  diagnosis.  The  tube-cultures  are  serviceable  in  giving  opportunity  for  a  further 
study  of  the  characters  as  well  as  to  furnish  the  most  convenient  method  of  maintain- 
ing the  cultures. 

GENERAL  DIRECTIONS.  Bacteria  when  ob- 
tained in  "pure  culture"  are  usually  grown  in 
test-tube  cultures.  To  make  these  a  small  portion 
of  a  previous  culture  is  transferred  to  fresh  cul- 
ture media  by  means  of  the  platinum  needles. 

a.  Stab  Cultures  are  made  in  test-tubes  con- 
taining solid,  transparent   media,  such  as   gelatin 
and  agar.     The  end  of  a  sterile  needle  is  infected 
with  the  material  to  be  transferred.     The  needle  is 
then  thrust  into  the  medium  to  the  bottom  of  the 
test-tube  and  withdrawn.     In  this  way  the  bacteria 
are  left  along  the  entire  length  of  the  needle  track. 
For  method  of  holding  tubes  see  fig.  7.     They  are 
held  in  an  inclined  position  to  prevent  the  possi- 
bility of  infection. 

b.  Streak  Cultures  are  cultures  made  by  drawing  the  needle  or  loop  over  the  surface 
of  the  medium  (test-tubes  with  media  having  sloped  surfaces  or  plate  cultures).     Agar, 
potato  and  blood  serum  are  frequently  used  in  this  way,  and  occasionally  gelatin. 

c.  Fluid  Cultures  (bouillon,  milk,  etc.),  are  inoculated  by  transferring  the  desired 
material  to  them  on  either  the  needle  or  loop. 

REFERENCES.     A.  146;  H.  51;  M.  &  E.  60;  McF.  146. 
SPECIAL  DIRECTIONS. 

a.  Make  a  gelatin  stab,  an  agar  streak,  a  potato  streak,  and  a  bouillon  culture  of 
Bacillus  subtilis  (EHRENB.)  COHN  (hay  bacillus)  and  Bacillus  coli  (EsCH.)  MIG.  (colon 
bacillus)  from  agar  cultures  supplied. 

b.  Label  each  tube,  writing  the  name  of  the  organism,  the  date  of  inoculation  and 
your  own  name. 

c.  Place  the  gelatin  in  the  cool  chamber,  and  the  other  cultures  in  the  incubator  at 
28°  C.     (XIV). 

EXERCISE  XIV.  INCUBATION  OF  CULTURES. 

EXPLANATORY.  Most  bacteria  grow  at  ordinary  temperatures  (22°  C.),  but  their 
growth  is  usually  hastened  by  a  higher  temperature  (e.  g.  28°-30°  C.).  The  pathogenic, 
or  disease-producing  bacteria  grow  best  at  the  temperature  of  the  human  body  (38°  0.). 
All  bacteriological  laboratories  are,  therefore,  supplied  with  apparatus  arranged  for 
maintaining  constant  temperatures,  known  as  thermostats  or  incubators. 

The  non-pathogenic  cultures  are  usually  kept  at  28°  C.,  while  the  pathogenic  ones 
are  kept  at  38°  C.  All  gelatin  cultures,  however,  must  be  kept  at  a  temperature  several 
degrees  below  the  melting  point  of  gelatin,  i.  e.,  not  above  22°  C.  Ordinarily  the  temper- 
ature of  the  locker,  especially  near  the  floor,  will  be  found  satisfactory.  In  a  very  warm 
room,  particularly  in  the  summer,  an  artificially  cooled  chamber  will  be  necessary. 

Test-tube  cultures  are  stored  in  the  various  incubators  in  tin  cans  or  glass  tumblers 
with  a  layer  of  cotton  in  the  bottom,  while  the  Petri  dishes  are  stacked  in  low  piles. 

REFERENCES.     A.  136;  H.  48;   M.  &  R.  88;  N.  178  &  243;   P.  231;  P.  &  M.  37. 


18  General  Bacteriology. 

SPECIAL  DIRECTIONS. 

a.  Incubate  all  cultures  of  the  non- pathogenic  bacteria  at  28°  C.,  except  the  gelatin. 
Keep  these  in  the  cool  chamber.    After  growth  has  taken  place,  the  cultures  can  be  taken 
from  the  incubator  and  kept  at  the  room  temperature. 

b.  Study  and  make  diagrams  of  an  incubator,  a  Reichert  thermo-regulator,  a  Roux 
thermo-regulator  and  Koch's  safety  burner. 

EXERCISE  XV.     CLEANING  SLIDES  AND  COVER-GLASSES. 

GENERAL  DIRECTIONS.  Slides  can  be  sufficiently  cleaned  by  washing  in  water  or 
alcohol  and  drying  with  a  towel.  The  cover-glasses  for  bacteriological  work,  however, 
must  not  only  be  freed  from  visible  dirt  but  must  be  rendered  free  from  fat.  One  of  the 
best  methods  is  the  following:  New  cover-glasses  are  cleaned  by  washing  in  water  and 
drying  from  alcohol  between  driers  (two  blocks  20xlOx2J-  mm.  covered  with  several  layers 
of  cotton  cloth  or  chamois  skin) ,  and  then  heating  them  on  a  piece  of  sheet  iron  or  in  hot 
air  sterilizer  for  one  hour  at  about  200°  C.  They  are  best  kept  in  a  clean  Petri  dish  and 
handled  with  forceps.  (Novy).  Old  slides  and  covers  having  balsam  on  them  should  first 
be  dropped  one  by  one  into  a  cleaning  solution  (potassium  bichromate  60,  sulphuric  acid 
60,  water  1000) ,  and  boiled  for  one-half  hour  and  then  treated  as  above. 

SPECIAL  DIRECTIONS.  Clean  £  oz.  of  cover-glasses  and  place  them  in  a  clean  Petri 
dish. 

EXERCISE  XVI.    PREPARATION  OF  STAINING  SOLUTIONS. 

GENERAL  DIRECTIONS.  The  dyes  most  useful  for  staining  bacteria  are  the  basic 
anilin  dyes  which  come  in  powdered  or  crystalline  form.  (Gruebler's  dyes  are  standard.) 
Those  in  most  common  use  are  Fuchsin,  Methylen  blue,  Gentian  violet  and  Bismark 
brown.  They  keep  well  in  powdered  form,  with  perhaps  the  exception  of  Methylen 
blue,  but  because  of  greater  convenience  and  equally  good  keeping  qualities,  saturated 
alcoholic  solutions  are  kept  in  stock.  These  are  made  by  adding  the  dry  dye  to  95% 
alcohol  to  saturation  and  filtering.  This  form  can  not  be  used  for  staining  bacteria.  The 
following  solutions  are  required  to  begin  work  with: 

1.  Aqueous  solution  of  Gentian  violet. 

Saturated  alcoholic  solution  of  Gentian  violet,  -      2.5  cc. 

Distilled  water,  47.5  cc. 

2.  Saturated  aqueous  solution  of  Bismark  brown. 

3.  Ziehl's  carbol-fuchsin. 

Saturated  alcoholic  solution  of  Puchsin.  5  cc. 

Solution  of  carbolic  acid  (5%)-     -  45  cc. 

4.  Loeffler's  Methylen  blue. 

Saturated  alcoholic  solution  of  Methylen  blue,  -      15  cc. 

Potassium  hydrate  ( 1 : 10 , 000 ) ,  50  cc . 

5.  Ehrlich's  Anilin  Oil  Gentian  violet. 

Saturated  alcoholic  solution  of  Gentian  violet,  6  cc. 

Absolute  alcohol,     -  5  cc. 

Anilin  water,  -  -  50  cc. 

Anilin  water  is  prepared  by  adding  2-3  cc.  of  anilin  oil  drop  by  drop  to  50  cc.  of 
water,  thoroughly  shaking  and  then  filtering  through  moistened  filter  paper  until  per- 
fectly clear. 


20 


General  Bacteriology . 


This  stain  should  stand  24  hours  and  then  be  filtered.  It  does  not  keep  well  and  must 


not  be  used  when  more  than  14  days  old. 

6.  Grain's  Iodine  solution. 

Iodine, 

Potassium  iodide, 
Distilled  water, 

7.  Gabbett's  Methylen  blue  solution. 

Methylen  blue  (dry), 
Sulphuric  acid, 
Distilled  water, 

8.  Alcohol,  96%. 

REFERENCES.  A.  156;  H.  75;  M.  &  W.  245;  M.  &  R. 
103;  McF.  90;  P.  200. 

SPECIAL  DIRECTIONS.  Prepare  the  solutions  of  dyes 
from  the  saturated  alcoholic  solutions  (furnished)  and 
place  them  in  2  oz.  bottles  arranged  with  pipettes  and  neatly 
labeled.  The  bottles  are  conveniently  kept  in  a  block. 
Fig.  8. 

EXERCISE    XVII.     SIMPLE    COVER-GLASS  PREPARATION. 


1  gm. 

2  gm. 
300  cc. 

2  gms. 
25  cc. 
75  cc. 


Block  for  stain  bottles. 


GENERAL  DIRECTIONS.  Bacteria  may  be  studied  under  the  microscope  in  a  living 
condition  in  a  hanging  drop  preparation  (XIX)  ;  but  on  account  of  their  hyaline  charac- 
ter, which  makes  the  examination  difficult,  the  student  should  first  learn  to  stain  them 
and  later  make  the  hanging  drop  preparation.  With  a  few  exceptions  all  bacteria  can 
be  stained  by  the  following  process  :  A  small  drop  of  distilled  water  is  placed  on  a  clean 
cover-glass  by  means  of  the  platinum  loop.  With  a  sterile  needle  a  portion  of  the  material 
to  be  examined  is  secured  and  while  the  cover-glass  is  held  in  the  fingers  of  the  left  hand 
the  bacteria  on  the  needle  are  introduced  into  the  water,  thoroughly  mixed  and  then 
spread  in  a  thin  film  over  as  much  of  the  surface  of  the  cover-glass  as  possible.  When 
the  bacteria  are  taken  from  fluid  media  a  drop  of  water  will  not  be  necessary.  In  this 
case  use  a  loop.  The  film  is  now  allowed  to  dry.  If  the  drop  is  sufficiently  small  this 
will  be  a  short  process.  It  may  be  hastened  by  holding  the  cover-glass  high  over  the 
flame,  but  it  should  always  be  held  in  the  hand  to  prevent  over-  heating,  which  spoils  the 
preparation. 

When  the  film  is  thoroughly  dry  place  the  cover-glass  in  a  pair  of  Cornet  or  Stewart 
forceps  and  "fix''  the  bacteria  in  the  flame.  This  is  done  by  passing  the  preparation 
through  the  upper  portion  of  a  gas  flame,  film  side  up.  Three  passages  should  be  made, 
each  consuming  about  one  second  of  time.  The  forceps  are  now  placed  on  the  table  and 
the  film  flooded  with  one  of  the  anilin  dyes.  After  the  stain  has  acted  for  five  or  ten 
minutes  it  is  washed  off  into  a  waste  dish  with  a  stream  of  distilled  water,  and  while 
the  cover-glass  is  still  wet  it  is  placed,  bacteria  side  down,  on  a  clean  glass  slide,  being 
careful  to  avoid  air  bubbles.  The  surplus  water  is  then  taken  up  by  means  of  a  small 
piece  of  blotting  or  filter  paper. 

The  preparation  is  now  ready  for  microscopical  examination.  (For  directions  see 
XVIII). 


22  General  Bacteriology. 

The  preparation  can  be  made  permanent  either  by  allowing  the  water  under  the 
cover-glass  to  dry  before  it  is  removed,  or  by  floating  it  off  with  water  and  afterwards 
drying.  When  dry  a  drop  of  Canada  balsam,  dissolved  in  xylene,  is  placed  on  the  cover- 
glass  and  this  is  then  lowered  on  to  the  slide  again. 

Resume . 

a.  Spread  film, 

b.  Air  dry, 

c.  Fix, 
(I.  Stain, 
e.  Mount, 
/.  Examine, 

g.  Mount  in  balsam,  or, 
/.  Mount  in  balsam, 
g.  Examine. 
"The  great  mistake  made  by  beginners  is  to  take  too  much  growth."  (M.  &  R.) 

REFERENCES.     A.  151;  H.  71;  L.  &  K.  104;  M.  &  W.  89;  M.  &  R.  95;   McF.  91; 
N.  147;  P.  198;   P.  B.  C.  11;   S.  25. 

SPECIAL  DIRECTIONS.  Make  cover-glass  preparation  from  agar  streak  of  B.  subtilis 
(XIII)  staining  with  au  aqueous  solution  of  gentian  violet  for  5  minutes. 

EXERCISE  XVIII.     USE  OF  MICROSCOPE. 

GENERAL  DIRECTIONS.  For  bacteriological  purposes  a  microscope  with  a  magnifying 
power  of  at  least  500  diameters  is  needed.  There  should  be  a  coarse  adjustment  (rack 
and  pinion)  as  well  as  a  fine  micrometer  screw;  and  the  following  accessories:  two  eye 
pieces,  one  1  in.  (25  mm.)  and  one  2  in. (50 mm.);  three  objectives,  one  1  in.  (16  mm.), 
one  i  in.  (4mm.),  ory  in.  (3.5  mm.)  and  one  oil  immersion  yV  in.  or  rV  in-  (2  mm.);  a 
triple  nose-piece,  and  an  Abbe  substage  condenser  with  iris  diaphragm  mounting. 

In  the  use  of  the  microscope  the  following  points  should  be  noted: 

a.  LIGHT.     The  proper  angle  at  which  the  mirror  should  be  placed  is  best  determined 
by  removing  the  eye-piece  and  so  arranging  the  mirror  that  the  unobstructed  light  from 
the  window  covers  the  whole  field.     The  ideal  light  is  that  from  a  white  cloud.     Direct 
sunlight  should  never  be  used. 

b.  ABBE  CONDENSER.  The  purpose  of  the  condenser  is  to  furnish  a  large  cone  of  light, 
and  as  it  is  corrected  for  parallel  rays  the  plane  side  of  the  mirror  should  always  be  used, 
except  when  artificial  light  is  employed.     When  highly  stained  objects  are  to  be  exam- 
ined, the  open  diaphragm  should  be  used,  but  when  the,  structural  rather  than  the  color 
picture  is  desired,  it  will  be  necessary  to  diminish  the  light  by  closing  the  diaphragm. 
When  the  high  powers  are  employed,  raise  the  condenser  as  high  as  possible;  for  low 
powers  a  lower  position  will  give  better  definition. 

c.  FOCUSING.     Turn  the  proper  objective  in  place  and  rack  down  until  the  objective 
nearly  touches  the  cover- glass.     This  should  be  done  while  the  eye  is  held  at  one  side  and 
directs  the  movement.     Then  with  the  eye  at  the  tube  slowly  move  up  with  the  micrometer 
screw-     Never  rack  down  with  the  eye  at  the  tube. 

d.  USE  OP  OIL- IMMERSION.  The  oil-immersion  objective  is  indispensable  to  the  proper 
study  of  bacteria.     It  is  constructed  upon  the  principle  that  a  drop  of  fluid  having  the 
same  refractive  index  as  the  objective,  prevents  the  dispersion  of  light,  thus  permitting 
the  use  of  lenses  having  a  greater  numerical  aperture  and  longer  working  distance  for 


24  General  Bacteriology. 

the  same  degree  of  amplification  than  is  possible  with  the  dry  system.  In  using  an 
immersion  lens,  place  a  small  drop  of  oil  on  the  preparation,  then  carefully  lower  the 
objective  until  it  touches  the  oil  drop  and  nearly  touches  the  cover-glass.  Apply  eye  to 
the  ocular  and  focus  upward  very  slowly  with  fine  adjustment  until  the  definition  is  clear. 
At  the  close  of  the  day's  work  the  oil  must  be  removed  from  the  objective  and  cover- 
glass.  This  is  best  accomplished  by  wiping  them  with  apiece  of  Japanese  paper  made  for 
the  purpose.  In  case  the  oil  should  accidentally  dry  on  the  objective,  it  can  be  removed 
by  adding  a  little  more  oil  and  allowing  it  to  stand  for  a  few  minutes;  it  can  then  be 
wiped  off  with  paper.  If  this  method  does  not  succeed,  the  objective  should  be  taken  to 
the  instructor.  Great  care  must  be  observed  since  solvents  of  the  oil  are  also  sol- 
vents for  the  lens  mountings. 

REFERENCES.     See  Gage;  A.  190;  H.  104;   M.  &  R.  93;  McF.  86;  N.  123;  P.  206. 

SPECIAL  DIRECTIONS. 

a.  Examine  cover-glass  preparations  made  in   (XVII)  first  with  \   in.  objective, 
and  then  with  the  oil-immersion  objective.     If  the  specimen  is  satisfactory  wipe  off  the 
oil  and  mount  in  Canada  balsam. 

b.  Practice  making  cover-glass  preparations  by  staining  specimens  from  each  of  your 
cultures.     Use  Loeffler's  methylen  blue  for  the  gelatin  and  bouillon;    aqueous  solution  of 
gentian  violet  for  agar,  and  carbol-fuchsin  for  potato.    Examine,  mount  permanently  and 
hand  to  instructor  for  inspection. 

EXERCISE  XIX.     HANaiNQ-DROP  PREPARATIONS. 

GENERAL  DIRECTIONS.  These  are  made  by  adding  a  small  portion  of  bacterial  cul- 
ture from  solid  media  to  a  drop  of  water  on  a  clean  cover-glass,  or  in  case  of  fluid  media 
by  placing  a  loop  of  the  culture  medium  on  the  cover- glass.  A  hollow  ground  glass 
slide  having  the  rim  of  the  cavity  previously  coated  with  vaseline,  is  inverted  and  lowered 
over  the  cover-glass  enclosing  the  drop.  With  a  careful,  quick  movement  the  prepara- 
tion is  now  brought  right  side  up. 

Instead  of  the  hollow  ground  glass-slide  an  ordinary  glass-slide  to  which  a  small 
section  of  a  glass  or  rubber  tube  has  been  cemented  can  be  used,  and  in  some  cases  is 
preferable. 

In  examining  the  preparation  under  a  microscope  focusing  is  a  somewhat  difficult 
process  and  must  be  carried  out  with  great  care.  Use  a  narrow  diaphragm.  Find  the 
edge  of  the  drop  with  the  low  power  (f  in.  objective)  adjusting  slide  so  that  edge  of 
drop  passes  through  the  center  of  the  field;  then  turn  on  the  high  power  (^  in.  objective) 
and  focus  without  moving  the  slide.  The  edge  of  the  drop  is  selected  because  the  bacteria 
are  here  nearest  the  cover-glass  and  hence  more  easily  focused  upon  than  where  they  are 
deeper  in  the  drop. 

REFERENCES.  A.  195;  H.  101;  L.  &  K.  102;  M.  &  W.  Ill;  M.  &  R.  94;  McF.  88; 
N.  142;  P.  209. 

SPECIAL  DIRECTIONS. 

a.  Make  hanging-drop  preparation  of  B.  subtilis  from  agar  or  bouillon.     (XIII) 

6.  Make  same  preparation  of  B.  coli.     (XIII) 

c.  Make  same  preparation  of  organism  supplied.     (Micrococcus) 

d.  Make  same  preparation  of  water  containing  particles  of  india  ink  or  carmine  in 
suspensiqn.     Study  character  of  movement  in  all  cases.     Distinguish  between  vital  and 
molecular  movement. 


26  General  Bacteriology. 

In  cases  where  vital  movement  is  questionable,  remove  the  cover-glass  and  place  a 
drop  of  formalin  or  chloroform  in  the  bottom  of  the  cell;  replace  the  cover-glass,  ex- 
amine and  note  change  in  character  of  movement,  if  any. 

EXERCISE  XX.    TEST-TUBE  CULTURES  ILLUSTRATING  FORM  TYPES. 

a.  Make  test-tube  cultures  in  bouillon,  gelatin,  agar  and  potato  of  the  following 
organisms : 

Micrococcus  (any  species). 

Sarcina  lutea  SCHROETEB. 

Pseudomonas  fluoresce ns  (FLUEGGE)  MIG. 

Bacillus  mycoides  FLUEGGE. 

Microspira  Metschnikovi  MIG.  (or  any  vibrio). 

Spirillum  rubrum  v.  ESMARCH. 

6.  Incubate  all  cultures,  except  gelatin,  at  28°  C. 

EXERCISE  XXI.     STUDY  OF  TEST-TUBE  CULTURES. 

GENERAL  DIRECTIONS.  As  soon  as  growth  becomes  visible  a  systematic  and  careful 
study  of  the  cultures  should  be  made.  A  detailed  list  of  the  points  to  be  noted  will  be 
found  in  Chapter  III,  and  should  be  consulted  in  writing  up  the  descriptions.  The  sum- 
mary below  will,  however,  be  found  useful. 

For  bouillon  cultures  note :  1)  condition  of  fluid,  2)  character  of  sediment,  3)  pres- 
ence or  absence  of  membrane,  and  4)  characteristic  odor. 

For  solid  cultures  (agar  and  potato  slopes),  note:  1)  Form  of  growth,  2)  size,  3) 
surface  elevation,  4)  consistency,  5)  color,  6)  effect  on  media,  and  7)  characteristic  odor. 

For  gelatin  stab  cultures,  note:  1)  Effect  on  media,  a.  non-liquefying,  i)  line  of 
puncture,  ii)  surface,  b.  liquefying,  i)  shape  of  liquefied  area,  ii)  condition  of  fluid,  iii) 
character  of  sediment,  2)  characteristic  odor. 

The  study  should  be  continued  from  day  to  day  as  long  as  changes  are  noted.  Make 
drawings  wherever  they  will  be  of  service  in  elucidating  the  descriptions. 

REFERENCES.     P.  B.  C.  (Charts  by  Cheesman.) 

SPECIAL  DIRECTIONS.  Study  and  write  careful  descriptions  and  make  necessary 
drawings  of  all  cultures  made. 

EXERCISE  XXII.     MICROSCOPICAL  STUDY  OF  FORH  TYPES. 

a.  Make  cover-glass  preparations  from  the  agar  streaks  (XX)  and  stain  with  an 
aqueous  solution  of  gentian  violet  or  with  Loeffler's  methylen  blue. 

b.  Examine  with  the  oil-immersion  objective,  write  the  names  of  the  organisms  in 
their  proper  place  in  the  table  below. 

Name.  Sketch. 


(  medium . 
Coccaceae  (spherical) 

(.  small . . 


( large . 
Bacteriaceae  (elongated) 

(  small . 


(  curved- . 
Spirillaceae  (spiral) 

(.  twisted . 


28 


General  Bacteriology. 


c.  Make  similar  preparations  from  the  gelatin  and  potato  and  note  any  variations  in 
form,  size.  etc. 

d.  All  these  preparations  are  to  be  mounted  in  balsam,    sketched   (XXIII)   and 
handed  to  instructor  for  inspection. 

EXERCISE  XXIII.     DRAWING  BACTERIA. 

GENERAL  DIRECTIONS.  In  drawing  bacteria  only  a  few  organisms  occuring  in  the 
microscopic  field  should  be  sketched,  but  these  should  be  made  of  considerable  size  so 
that  the  exact  outline  maybe  indicated.  Furthermore  they  should  be  drawn  to  scale  and 
individuals  selected  to  give  range  in  form  and  size. 

To  measure  microscopic  objects  an  ocular  micrometer  is  used,  and  the  first  step  will 
be  to  determine  its  value.  Place  the  ocular  micrometer  on  the  diaphragm  in  the  ocular, 
use  a  stage  micrometer  as  an  object  and  focus.  The  image  of  the  scale  on  the  stage  mi- 
crometer will  appear  imposed  on  that  of  the  ocular  micrometer.  Make  the  lines  of  the 
two  micrometers  parallel  and  then  make  any  two  lines  of  the  stage  micrometer  coincide 
with  any  two  on  the  ocular  micrometer,  pulling  out  the  draw-tube  if  necessary.  Divide 
the  value  of  the  included  space  or  spaces  on  the  stage  micrometer  by  the  number  of 
divisions  on  the  ocular  micrometer  required  to  include  them,  and  the  quotient  so  obtained 
will  give  the  valuation  of  the  ocular  micrometer  in  fractions  of  the  units  of  measure  of 
the  stage  micrometer  (Gage).  If  result  is  not  in  terms  of  micron  (//)  it  should  be  con- 
verted to  such,  as  this  is  the  unit  in  micrometry. 

In  making  drawings  represent  a  micron  by  two  millimeters  on  paper.  This  will  give 
a  magnification  of  2,000  (  X  2,000) . 

REFERENCES.     G.  100-108. 

SPECIAL  DIRECTIONS. 

a.  Determine  the  value  of  the  ocular  micrometer  and  fill  out  blanks  in  following 
table: 


No   of  Microscope  

Make  .  .  . 

Ocular  in 

.,  or  mm. 

Objective. 

Tube  length. 

Value  of  single  di- 
vision on  scale 
in  /*. 

|  in.  (16  mm.) 

^  in.  (4  mm.) 

Oil-immersion. 

6.  Make  drawings  of  cover- glass  preparations  made  in  XXII  in  place  provided  in 
table. 


30  General  Bacteriology. 

EXERCISE  XXIV.     STUDY  OF  CELL  GROUPING. 

HANGING  DROP  PREPARATIONS. 

a.  Make  hanging-drop  preparations  from  bouillon  cultures  prepared  above  and  also 
from  those  supplied. 

b.  Examine  with  oil-immersion  objective  and  assign  organisms  to  their  proper  place, 
as  determined  by  cell  grouping,  in  the  following  scheme : 

Name.  Sketch. 


Isolated 

(  Bacilli 
Filaments    • 

(.  Cocci 


Plane  surface,  Tetrads         

C  Regular  

Masses         -j  Irregular 

I 

I  Zoogloea         

IMPRESSION  PREPARATIONS.  The  exact  relation  of  cell  to  cell  as  they  develop  in  the 
colony  can  frequently  be  determined  with  greater  accuracy  by  studying  a  "contact  prep- 
aration" which  is  prepared  as  follows: 

a.  Melt  a  gelatin  tube  and  slope  it,  when  solid  make  a  streak  culture  of  B.  mycoides 
FLUEGG.E  and  when  growth  has  taken  place  dip  the  tube  in  hot  water  to  loosen  gelatin 
which  is  then  slipped  out  of  the  tube. 

b.  Lower  gently  a  clean  cover-glass  over  the  surface.     Apply  a  slight  pressure  by 
tapping  glass.     Raise  coyer-glass  by  one  edge  taking  care  that  natural  arrangement  of 
adherent  bacteria  is  not  disturbed. 

c.  Thoroughly  air  dry  the  same,  then  fix  and  stain  in  the  ordinary  manner. 

d.  Examine  the  thinner  layers  noticing  the  arrangement  of  cells  with  reference  to 
each  other  and  draw  a  sufficient  number  to  illustrate  this  relationship. 

AGAR  HANGING-DROP  CULTURES. 

a.  Melt  a  tube  of  agar  and  cool  to  43°  C. 

b.  Sterilize  a  cover- glass  by  passing  it  two  or  three  times  through  the  flame  quickly. 

c.  With  the  needle  make  a  streak  on  the  cover-glass  about  3  mm.  long  of  B.  subtilis. 

d.  With  the  loop  place  a  drop  of  liquid  agar  so  as  to  cover  up  streak. 

e.  Seal  cover-glass  to  hollow  ground  slide.     Incubate  and  later  examine  and  sketch. 

EXERCISE  XXV.    STUDY  OP  INVOLUTION  FORMS. 

a.  Grow  Bacillus  subtilis  (EHRENB.)     MIG.  in  bouillon  and  also  in  a  solution  con- 
taining 0.1%  asparagin,  10%  sugar,  and  by  means  of  stained  cover- glass  preparations 
compare  the  individual  organisms  in  each  casein  regard  to  their  form  and  size.     The  de- 
generated or  involution  forms  are  more  apparent  by  staining.      Draw  several  cells  illus- 
trating a  variety  of  involution  forms. 

b.  Examine  a  culture  of  Bacterium  diphtheriae  (LOEFFLER)  MIG.  on  Loeffler's  blood 
serum.     Read  M.  &  R.  5. 


32 


General  Bacteriology. 


EXERCrSE  XXVI.  GELATIN  PLATE  CULTURES. 

EXPLANATORY.  Plate  cultures  are  only  possible  with  the  liqueflable  solid  media, 
gelatin  and  agar.  In  making  them  the  bacteria  are  mixed  with  the  medium  while  it  is 
in  a  fluid  state  in  such  quantities  that  the  individuals  are  separated  from  each  other  by 
several  millimeters  when  it  is  spread  out  on  a  horizontal  surface  to  cool.  As  the  medium 
solidifies,  the  organisms  become  fixed  and  their  growth  results  in  the  formation  of  "colo- 
nies." These  vary  in  size  and  appearance  according  to  the  peculiarities  of  the  organism 
and  the  age  of  the  culture,  but  are  of  the  greatest  service  in  the  study  and  identification 
of  the  various  species.  These  cultures  are  prepared  as  follows: 

GENERAL  DIRECTIONS.  Three  gelatin  tubes  are  marked  Nos.  1,  2  and  3  and  melted 
by  placing  them  in  a  water  bath  at  a  temperature  of  42°  C.  For  this  purpose  a  small 

cup  of  water  placed  on  a  tripod  can  be  used  (Fig.  9).  They  are 
inoculated  by  introducing  the  material  to  be  studied  into  tube 
No.  1.  The  quantity  of  this  material  varies.  The  amount  cling- 
ing to  the  platinum  needle  will  be  sufficient  if  a  pure  culture  is 
used,  while  in  other  cases  several  loops  or  even  drops  are  neces- 
sary. The  inoculated  material  is  thoroughly  mixed  with  the 
gelatin  in  No.  1.  This  is  done  by  rolling  the  tube  gently  be- 
tween the  palms  of  the  hands,  instead  of  shaking,  so  as  to  pre- 
vent the  introduction  of  air  bubbles.  With  a  sterile  loop  three 
loopfuls  of  fluid  gelatin  are  now  transferred  from  No.  1  to  No.  2, 
and  mixed.  For  method  of  handling  tubes  see  Fig.  7.  In  like 
manner  three  or  more  loops  from  No.  2  are  carried  over  to  No. 
3,  which  in  turn  is  well  mixed.  The  cpntents  of  the  tubes  Nos. 
1-3  are  now  poured  into  separate  sterile  Petri  dishes. 
The  process  of  pouring  is  performed  as  follows:  The 
Petri  dish  is  placed  on  the  desk;  the  gelatin  tube  is 
taken  in  the  right  hand,  the  cotton  plug  removed  with 
the  left  hand;  the  mouth  of  the  tube  sterilized  by 
flaming  it  once  or  twice,  and  when  the  glass  is  cool  FIG.  10.  Method  of  pouring  plates, 
the  gelatin  is  poured  into  the  lower  half  of  the  dish  while  the  cover  is  slightly  raised 
(Fig.  10),  but  not  inverted  or  laid  on  the  table.  The  cover  of  the  dish  is  then  replaced, 
the  test-tube  filled  with  a  solution  of  corrosive  sublimate,  and  the  cotton  plug  returned. 
The  gelatin  is  spread  over  the  entire  bottom  of  the  dish  by  tipping  it  from  side  to  side. 
It  is  then  allowed  to  harden  by  placing  the  dish  on  the  cooling  apparatus  or  leaving  it 
on  horizontal  surface  at  room  temperature.  A  simple,  inexpensive  and  effective  cooling 

apparatus  is  a  piece  of  soapstone,  such  as  is  sold  at 
hardware  stores  (Fig.  11).  In  winter  this  can  be  cooled 
by  hanging  it  out  of  doors,  at  other  seasons  by  im- 
mersing it  in  cold  water.  The  three  Petri  dishes  thus 
prepared  should  be  properly  labeled  and  placed  un- 
der conditions  where  the  gelatin  will  remain  solid  and 
yet  growth  takes  place.  The  temperature  of  the 
laboratory  should  not  be  allowed  to  exceed  23°  C.  or 
gelatin  cultures  are  in  danger  of  melting  while  under  examination.  Within  a  few  days 
colonies  will  make  their  appearance,  in  varying  numbers,  depending  upon  the  dilution 
used. 


FIG.  9.  Method  of  melting  gela- 
tin. 


FIG.  11.    Soapstone  used  for  solidifying  gela- 
tin in  Petri  dishes. 


34 


General  Bacteriology. 


X 


Inasmuch  as  the  first  plate  is  invariably  too  thickly  seeded  to  be  of  much  service, 
this  gelatin  tube  is  often  replaced  by  a  water  blank,  which  is  treated  exactly  as  the  gela- 
tin tube  No.  1,  but  is  not  of  course  "plated"  but  simply  serves  to  dilute  the  material. 

REFERENCES.  A.  124;  H.  57;  L.  &  K.  88;  M.  &  W.  108;  M.  &  R.  61;  McF.  140; 
171;  P.  224;  S.  72. 

SPECIAL  DIRECTIONS. 

a.  Make  three  gelatin  plate  cultures,  as  directed  above,  and  inoculate  with  B.  sub- 
introducing  a  minute  portion  of  agar  culture  (XIII)  into  tube  No.  1,  two  loops  of 

No.  1  into  No.  2,  and  three  of  No.  2  into  No.  3.     Label,  and  when  the  gelatin  has  solidi- 
fied, place  plates  in  cool  chamber  (XV). 

b.  Also  make  a  "blank"  plate  from  an  uninoculated  gelatin  tube,  observing  all  pre- 
cautions to  prevent  contamination.     This  will  serve  as  a  control  or  check  on  your  other 
plates.     If  any  colonies  develop  on  this  it  indicates  carelessness. 

EXERCISE  XXVII.     AQAR  PLATE  CULTURES. 

GENERAL  DIRECTIONS.  These  are  made  in  the  same  way  as  the  gelatin  plates  ex- 
cept that  the  high  meltingpoint  (96°  C.)  of  agar  makes  it  necessary  to  use  boiling  water 
to  melt  it.  Inasmuch  as  the  vitality  of  vegetative  bacteria  is  destroyed  at  a  temperature 
much  above  42°  C.,  it  must  be  cooled  down  before  inoculating,  but  as  agar  solidifies  at 
39-40°  C.  it  must  not,  therefore,  be  cooled  below  that  point.  It  is  best  to  keep  the  melted 
agar  at  about  42°  C.  for  10  minutes  before  it  is  inoculated.  For  this  purpose  a  water- 
bath  should  be  so  arranged  that  the  temperature  can  be  controlled 
by  means  of  a  thermo-regulator.  A  cheap  and  yet  satisfactory 
03  W  M  ITl  ^  arrangement  is  represented  in  Fig.  11.  Inoculate,  make  dilu- 
V\  1  tions  and  pour  as  in  case  of  gelatin,  except  that  before  the  agar 

is  poured,  it  is  well  to  slightly  warm  the  Petri  dishes  by  placing 
them  iu  the  incubator  at  38°  C.  for  a  few  minutes,  other- 
wise the  agar  may  solidify  in  lumps  in  the  plate.  In  cooling, 
agar  shrinks  somewhat,  and  in  doing  so  water  is  expressed  from 
the  solid  jelly.  In  the  incubator  this  condenses  on  the  under 
side  of  the  cover  of  the  Petri  dish  to  such  an  extent  that  drops 
run  down  on  to  the  culture  surface  thus  causing  the  developing 
superficial  colonies  to  "run."  To  obviate  this  the  Petri  dishes, 


FIG.  11.    Water-bath  for  cooling 
agar. 


when  placed  in  the  incubator,  should  be  inverted. 
REFERENCES.     H.  61;  L.  &  K.  94;  M.  &  R.  66;   N.  285;   P.  225;   P.  B.  C.  28. 
SPECIAL  DIRECTIONS,     a.  Make  three  agar  plates  of  B.  coli;  use  one  loop  of  bou- 
illon culture   (XIII)  for  tube  No.  1  and  proceed  as  in  XXVI.     b.  Place  in  incubator  at 

EXERCISE  XXV11I.     ROLL-CULTURES  (Esmarch). 

GENERAL  DIRECTIONS.     These  are  essentially  plate  cultures  in  which  the  medium 
instead  of  being  poured  out  into  dishes  is  solidified  in  a  thin,  even  layer  on  the  inner  surface 

of  the  test-tubes.  This  is  best  accomplished  by  means  of  a 
piece  of  ice  placed  in  a  dish  on  a  piece  of  cloth  by  which  it 
can  be  kept  in  the  desired  position  (Fig.  12).  A  horizontal 
groove  is  melted  in  the  ice  by  means  of  a  test-tube  filled  with 
hot  water.  In  this  groove  the  test-tubes,  inoculated  as  in  case 
cuUu  '""of  plate  cultures,  are  rapidly  whirled  until  the  medium  is  thor- 

oughly set.     Both  agar  and  gelatin  can  be  used,  although  gelatin  cannot  be  used  sue- 


FIG. 12. 


36  General  Bacteriology. 

eessfully  with  those  species  which  liquefy  this  medium.  In  the  case  of  agar  the  tubes 
should  be  placed  in  a  horizontal  position  a  few  hours  (over  night)  until  the  medium  has 
become  attached  to  the  tube ;  afterwards  they  can  be  stored  in  the  usual  receptacles  for 
tube  cultures. 

REFERENCES.     A.  131;  M.  &  R.  65;   McF.  143. 

SPECIAL  DIRECTIONS.  «.  Melt  a  tube  of  gelatin  and  without  inoculating  it  practice 
making  a  roll-culture  as  described  above.  Avoid  tipping  the  tube  enough  to  get  medium 
on  cotton  plug.  Remelt  and  roll  again  until  the  knack  is  acquired. 

b.  Make  two  roll-cultures  in  gelatin  of  B.  coli  (XIII),  using  a  water-blank  instead 
of  gelatin  tube  No.  1. 

c.  Make  two  agar  cultures  of  B.  subtilis  in  same  way. 

d.  Incubate  b.  in  cool  chamber,  andc.  at  28°  C. 

EXERCISE  XXIX.     STUDY  OF  PLATE  CULTURES. 

MACROSCOPIC.  As  the  colonies  appear,  note:  «.  form,  b.  size,  c.  surface  elevation, 
d.  consistency,  t.  color.  Both  the  surface  and  deep  colonies  should  be  described  as  they 
are  frequently  very  different.  Drawings  should  always  be  made  wherever  they  will  be  of 
value;  study  should  be  continued  as  long  as  changes  are  noticed.  (See  Chapter  III, 
I.  A.  a.-f.) 

MICROSCOPIC.  The  colonies  appearing  on  the  plates  are  to  be  studied  under  a  low 
power  of  the  microscope.  Use  a  f  in.  (16  mm.)  objective.  The  Petri  dishes  can  be 
inverted,  and  thus  avoid  the  danger  of  exposing  the  culture  to  contamination  from  the 
air  except  with  gelatin  where  liquefying  organisms  are  present.  Observe,  «.  structure  of 
colony  as  a  whole ;  6.  character  of  margin.  (See  Chapter  III.  I.  A./ifcgr.) 

REFERENCES.     P.  B.  C.  (Cheesman's  Charts.) 

SPECIAL  DIRECTIONS.  Study,  write  descriptions  and  make  drawings  of  all  plate 
cultures.  Use  blank  pages  for  description  and  sketch  of  cultures. 

EXERCISE  XXX.     USE  OF  DECOLORIZING  AGENTS. 

Make  three  cover-glass  preparations  from  a  24  hour  old  culture  of  B.  subtilis,  stain- 
ing them  with  an  aqueous  solution  of  gentian  violet.  Mount  in  water  and  examine. 
While  they  are  still  under  the  microscope,  place  at  one  side  of  the  cover-glass  a  few 
drops  of  one  of  the  following  solutions,  and  by  means  of  a  strip  of  filter  paper  at  the 
opposite  side  draw  the  liquid  under  the  cov  er-glass  until  all  the  color  is  removed.  In 
this,  way  determine  the  relative  value  of  alcohol  (95%),  acetic  acid  (5%),  and  nitric  acid 
(30%)  as  decoloring  agents. 

EXERCISE  XXXI.     GRAM'S  STAIN. 

EXPLANATORY.  This  is  a  differential  stain  and  one  of  the  most  useful.  Some  bac- 
teria when  stained  by  this  method  exhibit  a  dark  violet  color,  others  remain  perfectly 
colorless,  thus  rendering  possible  the  differentiation  of  bacteria  which  are  morphologically 
nearly  or  quite  identical,  and  also  greatly  facilitating  the  demonstration  of  certain  bac- 
teria in  animal  tissue.  Most  of  the  pathogenic  micrococci  retain  the  violet  stain  although 
there  are  important  exceptions.  The  bacilli  and  spirilla  may  or  may  not  remain  colored. 

GENERAL  DIRECTIONS. 

a.  Spread  film. 

6.  Air  dry  and  fix. 


38  General  Bacteriology. 

c.  Stain  with  anilin-oil  gentian  violet  5  minutes. 

d.  Pour  off  stain  and  without  washing: 

e.  Apply  iodine  solution  2  minutes  (use  several  changes). 

/.  Decolorize  with  96%  alcohol  until  drippings  do  not  stain  white  filter  paper. 
g.  Wash  in  water  and  counter-stain  with  Bismarck  brown. 
h.  Mount  in  water  and  examine. 
i.   Dry  and  mount  in  balsam. 

REFERENCES.  A.  162;  H.  78;  L.  &  K.  106;  M.  &  W.  91;  M.  &  R.  110;  McF. 
99;  N.  287;  P.  203. 

SPECIAL  DIRECTIONS.  Stain  films  of  young  cultures  of  B.  coli  and  B.  subtilis.  Also 
a  film  of  an  organism  supplied. 

EXERCISE  XXXII.    TUBERCLE  STAIN  (Qabbett). 

EXPLANATORY.  All  of  the  differential  methods  of  staining  the  tubercle  bacterium 
depend  upon  the  fact  that  this  germ  is  very  resistant  towards  the  ordinary  stains  and  in 
order  to  be  stained  at  all  must  be  treated  with  a  dye  containing  a  mordant  and  this 
either  allowed  to  remain  in  contact  with  the  micro-organism  several  hours  or  be  applied 
hot.  The  latter  method  is  the  quicker  and  is  usually  employed,  although  it  does  not 
give  as  good  results.  When  once  stained  this  germ  withstands  the  effect  of  decoloriz- 
ing agents  to  such  an  extent  that  it  is  possible  to  remove  the  dye  from  all  other  objects 
on  the  cover-glass  preparation  (as  in  sputum)  while  it  retains  its  own  color.  The  appli- 
cation of  a  second  dye,  of  a  complementary  color,  readily  distinguishes  this  germ  from 
all  others  in  the  field.  A  few  other  bacteria  have  similar  staining  properties.  (See 
Part  II.)  Red  is  the  usual  stain  and  blue  the  counter  stain.  Gabbett's  method  is  one  of 
the  simplest. 

GENERAL  DIRECTIONS. 

a.  Spread  film  (sputum  from  tuberculous  patient) . 

b.  Air  dry  and  fix. 

c.  Stain  with  hot  carbol-fuchsin  2  minutes. 

d.  Wash  in  water. 

e.  Treat  with  Gabbett's  solution  |  to  1  minute. 
/.  Wash  in  water  and  examine. 

g.  Dry  and  mount  in  balsam. 

REFERENCES.     A.  162;  M.  &  W.  92;  McF.  214;  P.  304. 

SPECIAL  DIRECTIONS.  Stain  three  samples  of  sputa  which  contain  varying  numbers 
of  the  tubercle  bacteria. 

EXERCISE  XXXIII.     STAINING  ENDOSPORES. 

GENERAL  DIRECTIONS. 
A.  Simple  stain. 

a.  Prepare  film  as  usual. 

6.  Fix  by  passing  through  flame  10  or  12  times  instead  of  3  times.  (This  prevents 
the  vegetative  portion  from  taking  the  stain) . 

c.  Stain  2-5  minutes  in  hot  carbol-fuchsin. 

d.  Mount  and  examine. 


40  General  Bacteriology. 

B.  Differential  stain  (Hauser's  method). 

a.  Make  cover-glass  preparation  of  a  spore-bearing  culture,  fix  and  stain  with  hot 
carbol-fuchsin  until  spores  are  thoroughly  colored.     This  must  be  determined  by  mount- 
ing in  water  and  examining  under  microscope. 

b.  Cautiously  decolorize  with  acetic  acid,  5%,  until  stain  is  removed  from  the  vege- 
tative portion  only.     This  to  be  determined  as  above. 

c.  Wash  in  water  and  counter- stain  with  methylen  blue. 

d.  Examine.     Crimson  spores  will  be  seen  in  blue  bacilli. 
REFERENCES.     Other  methods,  see  A.  164-167. 

SPECIAL  DIRECTIONS.     Stain  by  each  method  spores  in  cultures  of  B.  subtilis  or 
other  spore-bearing  organisms. 

EXERCISE  XXXIV.     STUDY  OF  ENDOSPORES. 

a.  Make  cultures  on  peptoneless  agar,  or  agar  to  which  a  few  drops  of   calcium 
hydrate  has  been  added,  of  the  following  organisms  and  incubate  at  28°  or  38°  C.  depend- 
ing upon  the  organisms : 

Bacillus  subtilis  (EHRENB.)  COHN. 

Bacterium  anthracis  (KOCH)  MIG. 

Bacillus  amylobacter  VAN  TIEGHEM  (or  any  clostridium  form). 

Bacillus  tetani  NICOLAIER  (or  any  "drumstick"  bacillus). 

Pseudomonas  erythrosporus  (CORN)  MIG. 

b.  When  the  cultures  are  48  hours  old  mount  films  without  staining,  examine  and 
note: 

1)  Form. 

2)  Size. 

3)  Color. 

4)  Power  to  refract  light. 

5)  Relation  to  mother-cell. 

(1)  Median  or  central. 

(2)  Intermediate. 

(3)  Terminal  or  polar. 

(4)  Clostridium  form. 

(5)  Drumstick  form. 

c.  Make  drawings. 

READ:     J.  H.   26;   L.  60;    L.  &  N.  76;   M.   &  R.  6;    N.  46;   P.  46;   P.  B.  C.   15; 

S.  114. 

EXERCISE  XXXV.    FLAQELLA  STAIN  (Bunge). 

GENERAL  DIRECTIONS. 

a.  Make  an  agar  streak  of  the  organism  to  be  stained. 

b.  After  18  to  24  hours,  by  means  of  the  platinum  needle,  remove  a  portion  of  the 
growth  (being  careful  to  avoid  the  culture  medium)  to  a  large  drop  of  tap  water  on  a 
perfectly  clean  cover-glass  (XV.)  and  allow  to  stand  5  minutes  rather  than  spread,  as 
there  is  less  danger  of  breaking  off  the  flagella. 

c.  Spread  carefully  2  or  3  loopfulsof  this  drop  on  each  of  several  clean  cover-glasses 
and  dry  at  room  temperature. 

d.  Fix  by  passing  the  cover- glass  through  the  top  of  the  flame  while  it  is  held  in  the 
hand,  not  in  the  forceps,  as  over  heating  will  injure  the  preparation. 


42  General  Bacteriology. 

e.  Flood  the  cover- glasses  thus  prepared  with  the  following  solution  (Mordant) : 
Liquor  ferri  sesquichlorfdi  diluted  with  distilled  water  1:20,  1  part;  saturated  aqueous 
solution  of  taunic  acid,  3  parts.  This  mixture  improves  with  age  but  should  be  filtered 
before  using.  Allow  to  act  1  minute. 

/.  Wash  in  water  and  dry  between  filter  paper. 

g.  Stain  with  hot  carbol-fuchsin  for  about  one  minute. 

h.  Wash  in  water,  dry  and  mount  in  balsam. 

REFERENCES.  M.  &  W.  103;  McF.  104;  P.  205.  Other  methods  M.  &  R.  115; 
McF.  101;  A.  167. 

SPECIAL,  DIRECTIONS.     Stain  B.  typhi  from  cultures  furnished,  also  try  B.  coli  and 

B.  subtilis. 

EXERCISE  XXXVI.    CAPSULE  STAIN  (Welch). 

GENERAL  DIRECTIONS. 

a.  Spread  film  without  the  use  of  water. 

b.  Air  dry. 

c.  Fix. 

d.  Apply  glacial  acetic  acid,  and  drain  it  off  immediately.     Do  not  wash  in  water. 

e.  Stain  with  anilin-oil  gentian  violet  (Ehrlieh)  which   is    to  be  renewed  several 
times  to  remove  acid. 

/.  Wash  in  1  to  2%  salt  solution. 

g.  Examine  in  salt  solution.     (Balsam  causes  capsule  to  shrink.) 
REFERENCES.     A.  163;  P.  203;  P.  B.  C.  13. 

SPECIAL  DIRECTIONS.  Use  pneumonic  ("rusty")  sputum,  or  blood  of  rabbit  in- 
fected with  the  pneumococcus. 


CHAPTER  II. 

PHYSIOLOGY  OF  BACTERIA. 


EXERCISE  XXXVII.    PREPARATION  OF  SPECIAL  MEDIA. 

The  following  media  will  be  necessary  for  the  work  outlined  in  this  chapter: 

a.  GLUCOSE  BOUILLON.      To  ordinary  bouillon  add  1%  glucose  (c.  P.),  tube  and 
sterilize  in  steamer,  not  in  autoclave,  2  test-tubes  and  2  fermentation  tubes. 

b.  GLUCOSE  GELATIN.     1%  glucose  (c.  P.),  tube  and  sterilize  in  steamer,  6  tubes. 

c.  GLUCOSE  AGAR.     1%  glucose  (c.  P.),  5  tubes. 

d.  LACTOSE  AGAR.     1%  lactose  (c.  P.),  2  tubes. 

e.  LITMUS  SOLUTION.     To  10  gms.  of  the  dried  material  add  500  cc.  of  distilled  water, 
digest  in  a  warm  place,  decant  clear  liquid  and  add  a  few  drops  of  nitric  acid  to  produce 
a  violet  color.     (Button).     Place  in  flasks  or  test-tubes  and  sterilize  in  steamer  three 
times,  1  tube. 

/.  DUNHAM'S  SOLUTION. 

Sodium  chloride        0.5  gm.   |  fi  u        u    n  .    dissolved   fllt       tube  and  steril. 

Peptone  (Witte)        1.    gms.  \      .       .  .   , 

Water  100.  )       ze'  4 

g.  NITRATE 'SOLUTION. 

Sodium  chloride    0.5  gm. 

Peptone  (Merk)     1       gms.  ) 

Potassium  nitrate  0.2  [-Filter,  tube  and  sterilize,  3  tubes. 

Water  1,000  j 

k.  LITMUS  MILK. 

1)  Freshly  separated  milk  (or  if  this  is  not  available,  new  milk  is  placed  in  a  sep- 
aratory  funnel  in  an  ice  chest  over  night  to  allow  the  separation  of  the  cream  and  milk 
then  drawn  off)  is  titrated  with  /jNaOH  and  rendered  slightly  alkaline  to  phenolphtha- 
lein  by  the  addition  of  rNaOH. 

2)  Litmus  solution  is  then  added  until  medium  is  faintly  blue. 

3)  Tube  and  sterilize  in  the  steamer  for  30-45  minutes  on  3  or  4  consecutive  days. 
During  the  summer  months  particularly  very  resistant  bacterial  forms  abound  in  the 
milk  so  that  it  is  necessary  to  increase  the  number  of  applications  or  length  of  exposure. 
The  efficiency  of  the  sterilizing  process  should  be  tested  by  placing  the  flasks  in  the  in- 
cubator for  several  days  to  see  if  anv  change  occurs,  2  tubes. 

In  addition  to  the  above  have  15  tubes  of  bouillon  (9  to  contain  exactly  10  cc.  XLI 
&  XLIV),  10  tubes  of  gelatin,  15  tubes  of  agar,  6  water-blanks  and  5  potato  tubes. 

EXERCISE  XXXVIII.     EFFECT  OF  REACTION  OF  HED1A  ON  GROWTH. 

GENERAL  DIRECTIONS. 

a.  Melt  6  tubes  of  gelatin  and  add,  under  aseptic  precautions,  to  three  of  them,  re- 
spectively, 0.1  cc.,  0.3  cc.,  and  0.5  cc.  of  a  normal  solution  of  hydrochloric  acid,  and  to 
the  other  three  the  same  amounts  of  a  normal  sodium  hydrate. 


46  General  Bacteriology. 

b.  Thoroughly  mix,  solidify  gelatin  in  ice  water  and  then  inoculate  (stab)    each 
tube  with  the  organism  to  be  studied,  making  a  control  culture  in  a  tube  of  neutral 
gelatin. 

c.  Incubate  at  18°  C.  and  note  the  effect  of  the  chemicals  on  the  rate,  amount  and 
character  of  the  growth. 

REFERENCES.     L.  &  N.  87;   McF.  46. 

SPECIAL  DIRECTIONS.     Use  B.  subtilis  and  B.  coli. 

EXERCISE  XXXIX.     EFFECT  OF  CONCENTRATION  OF  MEDIA  ON  GROWTH. 

a.  Pour  about  2  cc.  of  "condensed  milk"  into  each  of  two  sterile  test-tubes,  dilute 
one  with  five  times  the  volume  of  sterile  water. 

b.  Inoculate    both    with    a   pure    culture    of    B.  subtilis  and  incubate  at   28°   C. 
Explain  changes  which  occur. 

c.  Test  extract  of  beef  or  syrup  in  the  same  way. 

EXERCISE  XL.     EFFECT  OF  TEMPERATURE  VARIATIONS  ON  RATE  OF  GROWTH. 

GENERAL  DIRECTIONS. 

a.  Make  four   agar  streak  cultures  of  organism  to  be  studied. 

b.  Incubate  them  at  the  following  temperatures:   Ice  chest  (7°  C.),  room  (20°  C.), 
low  incubator  (28°  C.),  blood  heat  (38°  C.). 

c.  By  frequent  observations  as  to  luxuriance  of  growth,  determine  the  optimum 
temperature  of  growth  for  each. 

REFERENCES.     F.  73;   L.  &  N.  98. 

SPECIAL  DIRECTIONS.     Use  B.  campestris  and  B.  coli. 

EXERCISE  XLI.     DETERMINATION  OF  THERMAL  DEATH  POINT. 

GENERAL  DIRECTIONS. 

a.  Make  a  bouillon  culture  of  the  organism  to  be  tested. 

b.  48  hours  later  heat  a  large  water-bath  to  45°  C.    Place  in  this,  in  close  proximity 
to  a  thermometer,  a  test-tube  (16  mtn.   in  diam.)    containing  exactly  10  cc.  of  standard 
bouillon.     (Reaction  +1.5.) 

c.  After  15  minutes  exposure  at  this  temperature  remove  the  cotton  plug  from  the 
tube,  inoculate  the  broth  with  three  loopfuls  (standard  size,  XII)  of  the  culture  prepared 
above  (a.),  and  carefully  mix  by  slightly  agitating  the  tube, without  removing  it  from  the 
bath. 

d.  After  a  further  exposure  of  10  minutes  remove  the  tube  from  the  bath  and  place 
it  in  a  vessel  of  ice  cold  water  to  cool.     Then  incubate  at  a  temperature  favorable  to  the 
development  of  the  organism  under  observation. 

e.  In  the  same  manner  expose  the  organism  to  the  following  temperatures:  50°,  55°, 
60°,  and  65°  C. 

/.  In  all  cases  incubate  at  least  a  week  and  take  as  the  thermal  death  point  the  low- 
est temperature  at  which  growth  fails  to  appear.     (In  more  accurate  work  the  tempera- 
ture should  be  determined  within  2°  C.). 
REFERENCES.     P.  B.  C.  32. 

SPECIAL  DIRECTIONS.      Use  B.  coli  or  B.  typhosus. 


48  General  Bacteriology. 

EXERCISE  XLH.     COHPARATIVE  EFFICIENCY  OF  DRY  AND  MOIST  HEAT. 

GENERAL  DIRECTIONS. 

a.  Charge  a  water  blank  with  culture  of  a  spore-bearing  bacillus,  shaking  it  well  to 
break  up  the  clumps. 

b.  Sterilize  eight  cover-glasses  by  passing  them  several  times  through  the  flame,  and 
place  four  in  each  of  two  sterile  Petri  dishes. 

c.  With  a  sterile  loop  place  an  equal  quantity  of  the  bacterial  suspension  (a.)  on 
each  cover-glass,  and  dry  by  placing  Petri  dishes  in  the  incubator  with  the  covers  slightly 
raised. 

d.  When  dry  place  one  Petri  dish  in  the  dry  sterilizer  (near  the  thermometer),  and 
the  other  in  the  steamer. 

e.  Keep  both  sterilizers  at    a  temperature  of  100°  C.,  and  at  the  end  of  5,  10,  20 
and  40  minutes  respectively,  remove  one  cover-glass  from  each  Petri,  place  it  in  a  sterile 
Petri  dish  and  pour  a  tube  of  liquefied  gelatin  or  agar  over  it.     Tip  the  dish  from  side 
to  side  to  dislodge  as  many  of  the  bacteria  as  possible  from  the  cover-glass,  solidify  the 
medium  and  incubate. 

REFERENCES.     L.  101;   S.  146. 

SPECIAL  DIRECTIONS.     Use  an  old  (spore-bearing)  culture  of  B.  subtilis.     Arrange 
data  in  the  form  of  a  table. 

EXERCISE  XLIII.     EFFECT  OF  DESICCATION. 

GENERAL  DIRECTIONS. 

a.  Prepare  five  cover-glasses  each  of  a  spore-bearing  and  a  non-spore-bearing  cul- 
ture, as  directed  in  XLII. 

b.  Place  them  in  a  sterile  Petri  dish,  and  dry  in  the  incubator. 

c.  Next  morning  and  every  twenty-four  hours  later  plate  one  of  the  cover-glasses. 

d.  In  this  way  determine  the  length  of  time  the  organism  in  question  can  withstand 
desiccation.  * 

REFERENCES.     F.  77;   L.  &  N.  93;  McF.  46;  S.  151. 

SPECIAL  DIRECTIONS.     Use  a  young  culture  of  B.  coli  and  an  old  (spore-bearing)  cul- 
ture of  B.  subtilis.     Tabulate  results. 

EXERCISE  XLIV.     EFFECT  OF  CHEMICALS  ON  BACTERIA. 

GENERAL  DIRECTIONS. 

a.  Inoculate  three  tubes  containing  10  cc.  of  sterile  bouillon,  with  three  loopfuls  of 
a  24-hour  old  broth  culture  of  organism  to  be  studied. 

b.  Add  0.1  cc.  of  a  5%  solution  of  carbolic  acid  to  one  tube  (No.  1);  0.6  cc.  to  an- 
other (No.  2) ;  and  2  cc.  to  the  third  (No.  3) . 

c.  Two  hours  later  transfer  three  loopfuls  from  each  tube  to  sterile  bouillon  and  in- 
cubate all  of  the  tubes  at  38°  C. 

d.  The  carbolic  acid  in  No.  1  and  its  sub-culture  does  not  prevent  growth.     In  No. 
2  no  growth,  but  abundant  in  its  sub-culture  (acts  as  an  antiseptic).    In  both  No.  3  and 
its  sub-culture  no  growth  (acts  as  a  disinfectant) . 

REFERENCES.     F.  81;  L.  &  N.  90;  L.  107;  McF.  46. 
SPECIAL  DIRECTIONS.     Use  B.  coli. 


50  General  Bacteriology. 

EXERCISE  XLV.    RELATION  TO  OXYGEN. 

GENERAL  DIRECTIONS. 

a.  Pour  a  tube  of  melted  agar  into  a  sterile  Petri   dish,    and  when  the  medium  has 
hardened  make  several  parallel  streaks  with  a  platinum  loop  charged  with  an  aerobic  or- 
ganism. 

b.  Sterilize  a  piece  of  mica  or  a  cover- glass,  by  passing  it  several  times  through  the 
flame  and  place  this  over  several  of  the  streaks.     This  is  to  shut  out  the  air  and  should 
therefore  be  in  perfect  contact  with  the  medium. 

c.  Make  another  plate  in  the  same  way  using  an  anaerobe. 
REFERENCES.     F.  60;  L.  &  N.  95;  L.  180;   McP.  Chap.  VIII. 
SPECIAL  DIRECTIONS.     Use  B.  siibtilis  and  an  anaerobe. 

EXERCISE  XLVI.     EFFECT  OF  DIRECT  SUNLIGHT. 

GENERAL  DIRECTIONS. 

a.  Make  an  agar  plate  of  the  organism  to  be  studied  (seeding  rather  thickly). 

b.  When  agar  has  thoroughly  set,  invert  the  Petri  and  paste  on  under  side  a  piece  of 
black  paper  from  which  has  been  cut  out  a  number  of  letters,  e.  g.,  student's  initials. 

c.  Expose  this  dish,  paper  side  up,   to  the  direct  sunlight  for  a  number  of  hours 
(4-6). 

d.  Remove  the  paper  and  incubate. 

REFERENCES.     F.  71;  L.  &N.  101;  L.  77;  McF.  46;  S.  151. 

SPECIAL  DIRECTIONS.     Use  B.  prodigiostis  (Ehrenb.)  Fluegge  or  B.  typhosus. 

EXERCISE  XLVII.    DETECTION  OF  GAS  (Shake  Culture). 

GENERAL  DIRECTIONS. 

a.  Melt   a  tube  of  glucose  agar  (or   gelatin)  and  inoculate  with   a  gas-producing 
organism. 

b.  Thoroughly  mix  and  solidify  quickly  by  placing  in  ice  water. 

c.  Incubate  over  night. 

REFERENCES.     L.  &  N.  153;  M.  &  R.,  85. 

SPECIAL  DIRECTIONS.     Use  B.  coli;  incubate.    Make  sketch. 

EXERCISE  XLVIII.    QUANTITATIVE  ANALYSIS  OF  GAS  (Fermentation  Tube). 

GENERAL  DIRECTIONS. 

a.  Inoculate  the  open  arm  of  a  fermentation  tube  with  a  gas-producing  organism. 

b.  Incubate  at  38°  C. 

c.  By  frequent  observations  determine: 

1.  Whether  growth  takes  place  in  the  open  or  closed  arm,  i.  e.,  whether  it  is  aero- 
bic or  anaerobic. 

^2.  The  rapidity  and  total  amount  of  gas  formation.  Use  gasometer.  (Plate  I.  B.) 
3.  Kinds  of  gas.  When  the  culture  has  ceased  producing  gas,  completely  fill  the 
open  arm  with  a  2  %  solution  of  sodium  hydrate ;  place  the  thumb  over  the  mouth  of  the 
tube  and  thoroughly  mix  the  Na  OH  with  the  gas  in  the  closed  arm,  then  without  remov- 
ing the  thumb  return  the  gas  to  the  closed  arm,  remove  the  thumb,  when  the  medium 
will  rise  in  the  closed  arm  to  take  the  place  of  the  absorbed  CO2.  Measure.  The  re- 


52  General  Bacteriology. 

maining  gas  is  considered  as  hydrogen;   bring  this  into  the  open  arm,  remove  the  thumb 
and  introduce  alighted  match.     Air  mixred  with  the  hydrogen  present  causes  a  slight  ex- 

TT 

plosion.  Express  the  amount  of  CO2  and  H.  in  the  form  of  a  proportion.     ^—  =    . 

CU2 

REFERENCES.     A.  203;  McF.  54;  M.  &  R.  86. 
SPECIAL  DIRECTIONS.     Use  B.  coli,  also  try  B.-subtilis. 

EXERCISE  XLIX.    DETECTION  OF  ACIDS  (Wurtz). 

GENERAL  DIRECTIONS. 

a.  Melt  a  tube  of  lactose  agar  (gelatin  can  be  used)  and  add  enough  of  a  sterile,  blue 
litmus  solution  to  give  it  a  distinct  color,  cool  to  42°  C.,  inoculate  it  with  an   acid- pro- 
ducing organism  and  pour  in  the  usual  manner. 

b.  When  the  agar  has  solidified  invert  the  dish  and  place  it  in  the  incubator. 
REFERENCES.     McF.  54. 

SPECIAL  DIRECTIONS.     Use  B.  coli  and  incubate  at  38°  C. 

EXERCISE  L.    QUANTITATIVE  DETERHINATION  OP  ACIDS. 

GENERAL  DIRECTIONS. 

a.  Inoculate    5    test-tubes  of    glucose   bouillon  (or   milk)  with   an  acid-producing 
organism. 

b.  At  periods  24  hours  apart  remove, with  a  sterile  pipette,  5  cc.  of  the  medium  from 
each  and  titrate  with  a  twentieth  normal  potassium  (or  sodium)  hydrate  solution,  using 
phenolphthalien  as  an  indicator. 

c.  Plot  the  results,  expressing  the  number  of  cc.  of  hydrate  solution  as  abscissae 
and  the  daily  intervals  as  ordinates. 

SPECIAL  DIRECTIONS.     Use  B.  coli  and  incubate  at  38°  C. 

EXERCISE  LI.    DETECTION  OF  NITRITES  IN  CULTURES. 

GENERAL  DIRECTIONS. 

a.  Make  a  culture  of  a  reducing  organism  in  a  test-tube  of  the   nitrate  solution 
(XXXVII.  g.). 

6.  Incubate  at  28°  C.  for  1  week,  add  1  cc.  of  each  of  following  solutions: 

1)  Sulphanilic  acid  (para-amido  benzenesulphonic  acid)   0.5  gm.     Acetic  acid  (sp. 
gr.  1.04)  150  cc. 

2)  «-amido-naphthalene   acetate.      Boil  0.1  gram  of  solid  a-amido-naphthalene  in 
20  cc.  of  water,  filter  the  solution  through  a  plug  of  washed  absorbent  cotton,  and  mix 
the  nitrate  with  180  cc.  of  diluted  acetic  acid.     All  water  and  vessels  used  must  be  free 
from  nitrites.     (Leffman  and  Beam.) 

The  presence  of  a  nitrite  is  indicated  by  a  pink  color. 

c.  A  tube  of  the  original  medium  should  be  incubated  and  tested  as  a  control. 

REFERENCES.     A.  215;  McF.  56. 

SPECIAL  DIRECTIONS.     Use  Bacillus  vulgaris.  (Hauser.)  Mig. 

EXERCISE  LII.     DETECTION  OF  AMMONIA. 

GENERAL  DIRECTIONS. 

a.  Make  bouillon  culture  and  incubate. 


54  General  Bacteriology. 

b.  Place  in  neck  of  tube  a  piece  of  filter  paper  which  has  been  dipped  in  Nessler's 
reagent  (for  formula  see  works  on  water  analysis).  A  yellow  to  reddish  brown  color 
indicates  the  presence  of  ammonia. 

REFERENCES.     L.  &  N. 141. 

SPECIAL  DIRECTIONS.     Use  sewage  to  inoculate  medium. 

EXERCISE  LIII.    DETECTION  OP  SULPHURETTED  HYDROGEN. 

GENERAL  DIRECTIONS. 

a.  Make  a  culture  in  a  test-tube,  or  better,  a  flask   of  bouillon  and  incubate  at 
38°  C. 

b.  Twenty-four  hours  later  fasten  in  the  flask,  by  means  of  the  cotton  plug,  a  strip 
of  filter  paper  moistened  with  lead  acetate. 

c.  The  presence  of  sulphuretted  hydrogen   is  indicated  by  change   of   color  from 
brownish  to  blue.     The  color  change  is  often  slight  and  can  be  best  detected  by  frequent 
observations. 

REFERENCES.     L.  &  N.  138. 

SPECIAL  DIRECTIONS.     Use  B.  coli  or  sewage. 

EXERCISE  LIV.     DETECTION  OF  INDOL. 

GENERAL  DIRECTIONS. 

a.  Make  a  culture  in  a  tube  of  glucose-free  broth*  (or  Dunham's  solution). 

b,  24  hours  to  1  week  later  add  a  few  drops  of  concentrated  sulphuric  acid  and  1  cc. 
of  sodium  nitrite  solution.     (Sodium  nitrite,  0.02  gms.      Distilled  water,  100  gms.) 

The  presence  of  iudol  is  indicated  by  the  production  of  a  deep  red  color. 
REFERENCES.     L.  &  N.  142;   McF.  56;   M.  &  R.  87. 
SPECIAL  DIRECTIONS.     Use  B.  coli. 

EXERCISE  LV.  DETERMINATION  OF  CHEMICAL  ENZYHES  IN  CULTURES. 

GENERAL  DIRECTIONS. 

a.  Make  two  gelatin   stab  cultures  of  a  rapidly  liquefying  organism   and  incubate 
several  days  or  until  the  gelatin  has  all  been  liquefied. 

b.  Pour  one  into  a  tube  of  gelatin   to   which  carbolic  acid    (yV  cc.  of  a  5%  sol.  per 
cc.  of  medium)  has  previously  been  added.     Mark  the  line  which  separates  the  liquid  and 
solid  gelatin. 

c.  Add  the  other  tube  of  liquefied  gelatin  to  a  tube  of  carbolized  milk. 

d.  Make  control  cultures  in  the  carbolic  media  with  a  pure  culture  of  the  organism 
used  above  to  show  that  the  acid  inhibits  the  growth  and  that  the  changes  are  not  due 
to  the  living  organism. 

REFERENCES.     McF.  53. 

SPECIAL  DIRECTIONS.     Use  B.  subtilis. 

EXERCISE  LVI.    VARIATION  IN  ENZYME  PRODUCTION. 

Make  stab  cultures  of  Pseudomonas  aeruginosa  (SCHROETER)  MIG.  (B.  pyocyaneus), 
or  any  slow  liquefier,  in  ordinary  neutral  gelatin  and  also  glucose  gelatin.  Compare 
rate  of  liquefaction  in  each. 


*This  is  prepared  from  beef  by  inosulating  the  meat  infusion  with  an  organism  capable  of  fer- 
menting sugar,  such  as  B.  coli,  and  allowing  it  to  stand  several  hours  at  !)8°  C.  The  meat  is  then 
strained  and  the  bouillon  prepared  in  the  usual  manner.  This  is  recommended  for  testing  for  indol. 


56  General  Bacteriology. 

EXERCISE  LV11.     VARIATION  IN  COLOR  PRODUCTION. 

Make  an  agar  streak  of  B.  prodigiosus.  Incubate  at  38°  C.  24  hours  later  transfer  to 
fresh  media.  Continue  the  process  of  daily  transplanting  from  cultures  of  previous  day 
until  chromogenic  property  is  lost,  even  at  the  room  temperature. 


CHAPTER  III. 

TAXONOMY. 

POINTS  TO  BE  OBSERVED  IN  THE  STUDY  OF  BACTERIA. 

The  following  scheme  gives  an  idea  of  the  points  to  be  noted  in  the  description  of 
an  organism  together  with  some  of  the  more  common  descriptive  terms. 

CULTURE  CHARACTERS. 
1.  GELATIN  PLATE: 

A.  Surface  colonies. 

a.  Form:   Punctiform,  too  small  to  be  defined  by  naked  eye;  circular;  oval; 
irregular;  fusiform;   cochlate,  twisted  like  a  snail  shell;    amoeboid,    very  irregular  like 
changing  forms  of  amoebae;  conglomerate,  an  aggregation  of  colonies. 

b.  Size,  expressed  in  millimeters. 

c.  Surface  Elevation:     flat;  spreading;  thin;  raised,  growth  thick  with  abrupt, 
terraced  edges;  convex,  surface  segment  of  a  circle  but  very  flatly  convex;  pulvinate,  sur- 
face the  segment  of  a  circle  but  decidedly  convex;  capitate,  hemispherical;  rough,  irregular 
elevations  and  depressions;  contoured,  like  the  undulating  surface  of  a  relief  map;  papil- 
late, horn  like  projections;    rugose,   wrinkled;    alveolate,   depressions  separated  by    thin 
walls;  pitted;  sulcate,  ridged  or  furrowed. 

d.  Consistency:      thin;  membraneous,  thin,  dry,  separating  from  medium;  coria- 
ceous, thick  like  leather  or  parchment;  viscous,  ropy;  slimy ;  gelatinous;  brittle. 

e.  Color:   transparent;    vitreous,  transparent  and  colorless;    oleaginous,    trans- 
parent and  yellow,  olive  to  linseed  oil  colored;  resinous,  transparent  and  brown,  varnish 
or  resin  colored;   translucent;  paraffinous,   translucent  and  white,  porcelaneous ;    opales- 
cent,    translucent,     grayish-white     by    reflected    light,    smoky-brown    by    transmitted 
light;     nacreous,    translucent,    grayish- white   with    pearly    lustre;     sebaceous,     trans- 
lucent,    yellowish     or     grayish- white,      tallowy;      butijrous,     translucent    or    yellow; 
ceraceous,     translucent   and  wax    colored;    opaque;    cretaceous,     opaque    and      white; 
chalky,  dull  without  lustre;  glossy,  shining;  fluorescent;  iridescent. 

f.  Margin  (To  be  determined  by   low  power  of  microscope):  entire;  undulate; 
repand;  erase,  finely  eroded  as  if  gnawed; lobed;  articulate;    laciniate,  cut  jaggedly  into 
deep  narrow  lobes;    lacerate,  cut  variously  into  irregular    segments'   fimbricate,  edge 
bordered  by  slender  processes  thicker  than  hairs;  ciliate,  tufted;  floccose,  wooly,  filaments 
in  fleecy  masses;  curled,  filaments  in  locks  or  ringlets:  filamentous,  consisting  of  loosely 
placed,  interwoven  filaments,  not  so  dense  as  floccose. 

g.  Internal  structure  (To  be  determined  by  microscope) :   homogeneous,  uniform 
throughout;   concentrically  zoned;  marmorated,  traversed  by  veins  as  in  some  kinds  of 
marble,  marbled;  finely  punctate;  areolate,  marked  out   with   small   spaces,  reticulate; 
moruloid,  having  the  character  of  a  morula,   resembling  a  mulberry;  segmented;  finely 
granular;  coarsely  granular;  grained,  as  in  lumber;  curled,  composed  of  twisted  bundles 
of  parallel  filaments  as  in  locks  or  ringlets;  floccose;  filamentous. 

h.  Change  in  Medium:   consistency;  color;  odor. 


58  General  Bacteriology. 

B.  Deep  colonies: 
a.  Form. 
6.  Size. 

c.  Color. 

d.  Internal  structure. 

2.  AGAB  PLATES: 

A.  Surface  colonies.  )  „  .    ,         .  ,.  ,.-, 

>  Same  points  as  in  gelatin  plate,  (I). 

B.  Deep  Colonies.       j 

3.  GELATIN  STAB  CULTURES. 

A.  Non-liquefying. 

a.  Line  of  puncture:  filiform,  uniform  growth  without  any  special  characters; 
tuberculate;  papillate,  covered  with  papilla?;   echinulate,  minutely  prickly;  villous,  beset 
with  long  or  short  undivided  hair-like  extensions;   arborescent,  beset  with  branched  hair- 
like  extensions;   beaded,   composed  of  small  round  more  or  less  conjointed  colonies; 
banded  longitudinally- 

b.  Surface:   (Same  as  surface  colonies  gelatin  plates  1  c.) 

B.  Liquefying. 

a.  Shape  of  liquefied  area:  crateriform,  saucer  shaped  liquefaction  of  gelatin; 
saccate,  shape  of  an  elongated  sack,  tubular;  cylindrical;  funnel  formed;  napiform,  out- 
line of  a  turnip;  fusiform,  outline  of  a  parsnip;  stratiform,  liquefaction  extending  to 
the  walls  of  the  tube  and  then  downward  horizontally. 

b-  Fluid:   clear;  turbid;  flocculenf. 

c.  Sediment:  flocculent;  stringy ;  granular. 

d.  Membrane:   character;  color. 

4.  STREAK  CULTURES: 

a.  Form. 

b.  Size. 

c.  Surface  elevation. 

d.  Consistency. 

P  i    .  f  Same  as  for  colonies  on  gelatin  plates  (1;. 

/.    Margin. 

g.  Internal  structure. 

h.  Change  in  medium- 

5.  POTATO. 

A.  Growth  apparent.    (Same  as  plate  cultures). 

B.  Growth  not  apparent. 

6.  BOUILLON: 

o.  Character  of  fluid :  clear;  turbid;  etc. 

b.  Sediment. 

c.  Membrane. 

7.  MILK. 

A.  No  visible  change,  even  after  boiling. 

B.  Curd  formed: 

a.  Time  required. 

b.  Character  of  curd:  hard;  soft. 

c.  Digestion- 


General  Bacteriology.  59 

d.  Character  of  whey:  clear;  turbid;  flocculent. 

e.  Reaction. 
/.  Gas. 

g.   Odor. 
8.  BLOOD  SERUM:      (Same  as  streak  cultures) . 

MORPHOLOGICAL   CHARACTERS. 

a.  Form. 

6.  Cell  grouping. 

c.  Size. 

1.  In  terms  of  the  inicromillimeter;    breadth,  average  and   extreme  length- 

2.  In  terms  of  human  blood  cell. 

d.  Stain. 

1.  Aqueous  solutions;   stains  easily  or  with  difficulty;  uniformly  or  irreg- 
ularly. 

2.  Special  stain ;   Gram;   tubercle;  etc. 

e.  Motility. 

1-   Brownian  movement- 

2.  Vital  movement;  sluggish  or  active;   rotary  or  direct;   most  favorable 
temperature;  age;  media;  etc. 

3-  Flagella;  stained  by  Loeffler,  Bunge  or  Van  Ermengem's  method;   dis- 
tribution, monotrichal,  lophotrichal  or  peritrichal- 

/.  Capsule;  stained  by  Ziehl,  Gram  or  Welch's  method;  most  favorable  con- 
ditions; broad  or  narrow;  present  in  serum,  milk  or  on  agar  streaks. 

g.  Spores;  time  required  for  formation;   media;  position  in  cell,  center  or  end; 
effect  on  shape  of  cell,  clostridium,  or  drumstick;  germination,  time,  temperature;  stain, 
Hauser  or  Moeller's  method;   temperature  limits- 
h.  Vacuoles  (plasmoloysis). 
i.  Crystals, 
j-  Involution  forms- 
k.  Pleomorphism- 

1.  Effect  of  various  media. 

2.  Effect  of  reaction  of  media- 

PHYSIOLOGICAL  CHARACTERS- 

a.  Effect  of  desiccation. 

b.  Relation  to  temperature;    minimum;    optimum;    maximum;    thermal  death 
point. 

c.  Relation  to  oxygen;  under  mica  plate;   in  hydrogen. 

d.  Relation  to  light;   (Buchner's  Experiment  XL VI.). 

e.  Relation  to  antiseptics  and  disinfectants • 

/-  Pigment  production;  relation  of  development  to  oxygen;  relation  of  de- 
velopment to  character  of  medium;  changes  produced  by  alkali  and  acid;  solubility;  spec- 
trum analysis. 

g-  Gas  production;  rate,  quantity  and  formula  produced  on  glucose,  lactose, 
and  saccharose  media. 


60  General  Bacteriology. 

h.  Acid  and  alkali  production;   carbohydrates  present ;   carbohydrates  absent, 
t.   Relation  of  growth   to  acidity  and  alkalinity  of  medium;    growth  in  1.5,  3 
and  4  %  alkali;   growth  in  1.5,  3,  4  and  5  %  acid. 

j.  Reduction  of  nitrates;   to  nitrites;   to  ammonia. 
k.  Production  of  sulphuretted  hydrogen. 
I-  Production  of  indol. 

m.  Enzyme  production;   proteolytic;   diastatic. 
n.  Characteristic  odor. 
o.  Pathogenesis : 

1.  Modes  of  inoculation  by  which  its  pathogenic  properties  are  demonstrated. 

2.  Quantity  of  material  required. 

3.  Duration  of  the  disease  and  its  symptoms- 

4.  Lesions  produced  and  the  distribution  of    the  bacteria  in  the  inoculated 

animals. 

5.  Which  animals  are  susceptible  and  which  are  immune. 

6.  Variations  in  virulence  and  the  probable  causes  to  which  they  are  due- 

7.  Detection  of  toxic  or  immunizing  products  of  growth. 
8-  Widal  test. 

9.  Pfeiffer's  phenomenon- 

REFERENCES:     Chester,  Report  Delaware  Experiment  Station,  1897;  A.  216;   P.  B. 
C.  (Cheesman's  Charts). 

CLASSIFICATION  OF  BACTERIA.     (MIQULA.) 

I.   Cells  globose  in  a  free  state,  not  elongated 
in  any  direction  before  divisions  in  1, 
2, or  3  planes.  COCCACEAE  ZOPH  emend.  MIG. 

A.  Cells  without  organs  of  motion. 

a.  Division  in  one  plane,  1.  Streptococcus  BILLROTH. 

b.  Division  in  two  planes,  2.  Micrococcus  (HALLIER)  COHN. 

c .  Division  in  three  planes,  •                    3.  Sarcina  Goodsir. 

B.  Cells  with  organs  of  motion. 

«.  Division  in  two  planes,      -  4.  Planococcus  MIGULA. 

b.  Division  in  three  planes,    •  5.  Planosarcina  MIGULA. 

II.  Cells  cylindrical,  longer  or  shorter,  and  only  di- 
vided in  one  plane,  and  elongated  to  twice  the 
normal  length  before  the  division. 

(1)  Cells  straight,  rod-shaped  without  sheath, 

non-motile  by  means  of  flagella.        BACTERIACEAE  MIGULA. 

A.  Cells  without  organs  of  motion,       -  6.  Bacterium  EURENB. 

B.  Cells  with  organs  of  motion  (flagella). 

a.  Flagella  distributed  over  the  whole 

body,  7,  Bacillus  COHN. 

b-  Flagella  polar,  8.  Pseudomonas  MIGULA. 

(2)  Cells  crooked,  without  sheath.  SPIRILLACEAE  MIGULA. 

A.  Cells  rigid,  not  snake-like  or  flexuous. 


General  Bacteriology.  61 

«.  Cells  without  organs  of  motion  (flag- 

ella),      -  9.  Spirosoma  MIGULA. 

b.  Cells  with  organs  of  motion  (flagella) 

1.  Cells  with    1,    very  rarely  2-3 

polar  flagella,.       -  -       10.  Microspira  SCHEOETER. 

2.  Cells  with  polar  flagella- tufts,       11.  Spirillum  EHRENB. 
B.  Cells  flexuous,       -  12.  Spirochaeta  EHRENB. 

(3)  Cells  enclosed  in  a  sheath.  CHLAMYDOBACTERIACEAE  MIGULA. 

A.  Cell  contents  without  granules  of  sulphur. 

a.  Cell  threads  unbranched. 

1) .  Cells  division  always  only  in  one 

plane,  -       13.  Streptothrix  COHN. 

2).  Cell  division  in  three  planes  pre- 
vious to  the  formation  of 
condia- 

i).   Cells  surrounded  by  very 
delicate   scarcely  visible 

sheath  (marine),   -         -  14.  Phragmidiothrix  ENGLER. 
ii).  Sheath  clearly  visible 

(fresh  water),    -         -       15.   Crenothrix  COHN. 

b.  Cell  threads  branched,  -       16.   Cladothrix  COHN. 

B.  Cell  contents  containing  sulphur  granules.  17.   Thiothrix  WINOGRADSKY 
(4).  Cells  destitute  of  a  sheath,  united  into  threads 

motile  by  means  of  an  undulating 
membrane.  BEGGIATOACEAE. 

Only    one    genus.     (The    single    species    is 

scarcely  separable  from  Oscillaria)  -  18.  Beggiatoa  TRAVISAN. 


CHAPTER  IV. 

SYSTEMATIC  STUDY  OF  REPRESENTATIVE  NON-PATHOGENIC 

BACTERIA. 


EXERCISE  LVIII.     PREPARATION  OF  SPECIAL  MEDIA. 

Tube  and  sterilize  the  following  media  for  work  in  Chapters  IV  and  V: 
80  tubes  of  plain  agar. 

2  tubes  of  lactose  agar. 
20  tubes  of  gelatin. 

8  tubes  of  bouillon. 
10  fermentation  tubes  of  glucose  bouillon. 

8  tubes  of  potato. 

8  tubes  of  milk. 

8  tubes  of  Dunham's  solution. 
10  water-blanks. 


EXERCISE  LIX.     BACILLUS  PRODIQIOSUS  (Ehrenb.)  Fluegge. 

EXPLANATORY.  This  organism  was  first  described  by  Ehrenberg  (Erhandlunger  der  Berliner  Akademie)  in  1839  and  named  Monas 
prodigiosa.  It  is  the  oldest  known  chromogenic  bacterium.  It  is  commonly  found  in  the  air  of  Europe  and  has  a  very  interesting  his- 
tory on  account  of  its  casual  relation  to  bread  epidemics — "bloody  bread,"  "bleeding  host,"  etc.  It  is  questionable  if  it  occurs  sponta- 
neously in  this  country.  It  is  slightly  pathogenic.  Introduced  intraperitoneally  into  guinea  pigs  in  large  quantities  it  produces  death. 
Inoculated  into  animals  naturally  immune  to  malignant  oedema  it  renders  them  susceptible.  Rabbits  inoculated  with  anthrax  are  protected 
by  a  subsequent  inoculation  with  this  organism.  It  is  grown  with  the  streptococcus  of  erysipelas  to  produce  Coley's  Fluid  for  treatment 
of  inoperable  malignant  tumors. 

REFERENCES.    Lafar,  137-138. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 
cultures. 

Incubatioa 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                  

2   Size                   ..          

5    Motility 

* 

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  ; 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as 

desiccation,  light,  disinfectants,  etc. : 

4.  Pigment  production: 


5.  Gas  produ  ction  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours ,  48  hours 

fecal  odor;  24  hou  rs ,48  hours 

9.  Enzyme  production  :  proteolytic 


....(s)    closed  arm:    

percent.,  72  hours  per  cent., hours  . 

...(5)    gas  formula  :  H  :  CO2  :  :  : 


per  cent. 


,  to  ammonia. 


.  days, 
days. 


.  diastatic. 


10.  Characteristic  odor. 

11.  Hathogenesis 


[64] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.(°C) 

34  HOURS. 

48        .     HOURS 

0             Dvrs 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

r 

A 

(4) 

Agar 
streak. 

^_  S 

A 

V                / 

A 

(5) 
Potato. 

Bouillon. 

(7) 

Special 

Media. 

[65] 


EXERCISE  LX.     VARIETY  OF  PIGMENTS. 

Make  agar  or  potato  streak  cultures  of  the  following  organisms,  incubate  at  38°  C..  study,  describe  and  sketch. 


AGAR  STREAK. 


24  HOURS. 


48  HOURS. 


SKETCHES. 


Bacillus 
indicus 
or 


Sarcina 
aurantiaca 
or 


Sarcina 
lutea 
or 


Pseudomonas 
fluorescens 
(B.  fluorescens) 


Pseudomonas 
aeruginosa 
(B.  pyocyaneus) 


Pseudomonas 
violacea 
or 


[66] 


General  Bacteriology.  67 

EXERCISE  LXI.    SEPARATION  OF  BACTERIAL  COLORING  HATTER. 

a.  Make  four  agar  streaks  of  Bacillus  prodigiosus,  which  are  to  be  kept  in  the  dark 
until  the  coloring  matter  is  well  formed. 

b.  Add  about  10  cc.  of  ether  to  each  tube  and  shake  vigorously  until  the  red  pig- 
ment has  all  been  dissolved  out. 

c.  Pour  into  a  large  test-tube  and  allow  to  stand  over  night  in  the  dark,  then 
pipette  off  the  colored  portion. 

d.  Divide  this  into  four  parts  and  treat  them  as  follows: 

1.  Evaporate  on  glass  slide  and  examine  crystals  formed  under  microscope. 

2.  Add  a  few  drops  of  hydrochloric  acid,  drop  by  drop. 

3.  Add  a  few  drops  of  sodium  hydroxide. 

4.  Stand  in 'direct  sunlight. 


EXERCISE  LXII.    BACTERlUn  PHOSPHORESCENS  Fischer. 

GENERAL  CONSIDERATIONS.  Described  by  Fischer  in  1887  (Zeitschrift  fttr  Hygiene,  Baud  II,  p.  92).  Found  in  Kiel  harbor,  dead 
sea  fish,  oysters  and  occasionally  on  meat  in  shops.  The  production  of  light  is  shown  in  the  dark,  especially  when  the  organism  is 
grown  on  a  medium  made  by  boiling  two  salt  herrings  in  a  liter  of  water,  adding  100  gins,  of  gelatin  to  the  filtrate  without  neutraliza- 
tion, tubing  and  then  sterilizing  (Lehmann).  Phosphorescence  can  even  be  restored  to  attenuated  cultures  by  growth  on  this  medium. 
Inasmuch  as  oxygen  is  necessary  to  light  production  surface  growths  are  best. 

REFERENCES.     Lafar  160-164. 


MORPHOLOGICAL  CHARACTERS. 

•I 

V 

3* 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                                                                        .                     ....                                                 

2   Size      

in  growths  

4.  Staining  powers:  

5.  Motility  *  

a.  Character  of  movement  

b.  Flagella  stain  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.    Relation  to  free  oxygen: 

5.    Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: . 
4.    Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

A.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours  . 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteoly tic 


. . . .  (2)    closed  arm :    

per  cent.,  72  hours per  cent., 

•  -.(5)    gas  formula,    H  :  CO2  :  : 


hours per  cent. 


.  to  ammonia. 


.  days, 
days. 


.  diastatic  — 


10.  Characteristic  odor. 

11.  Pathogenesis 


[68] 


CULTURE  CHARACTERS. 


Kc'art'um 
of  Medium. 
Incubation 
Temp.  (°C) 


24 


HOURS. 


48. .      .  HOURS. 


6..      .  DAYS. 


SKETCHES. 


[69] 


A 


EXERCISE  I. Mil.     BACILLUS  ACIDI  LACTIC!  Hueppe. 

GENERAL  CONSIDERATIONS.     First  described  in  1884  by  Hueppe  in  Mitteil.  aus  dem  Kaiserl.  Gesundlieitsamte,  Bd.  II,  p.  1837.     This 
organism  may  be  taken  as  a  type  of  the  bacteria  causing  sour  milk. 
REFERENCES.    Lafar,  223-244. 


MORPHOLOGICAL  CHARACTERS. 

• 

v 

~°s 

o'a 

*u 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin 

2   Size  

5    Motility                                                                                                                                

b   Flagella  stain                                    .                                                                  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: '. 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

4.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


...(2)  closed  arm: 

.  per  cent.,  72  hours per  cent., 

...(5)    gasformula,  H  :  CO,  :  : 


—  hours.. 


percent. 


to  ammonia.. 


days, 
days. 


.diastatic. 


10.    Characteristic  odor., 
it.    Pathogenesis 


[70] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium. 
Incubation 
Temp.  CC) 


24..     ..HOURS. 


48 HOURS. 


6 DAYS. 


[71] 


SKETCHES. 


, 


A 

i 


A 


EXERCISE  LX1V.    BACILLUS  VULQARIS  (Hauser)  Migula. 

PROTEUS  VULGARIS. 

GENERAL  CONSIDERATIONS.  Described  by  Hauser  in  1885  as  Proteus  vulgaris  (Ueber  Faulnis  Bakterien).  It  is  widely  distributed 
and  is  commonly  found  in  putrefactive  substances.  It  is  one  of  several  related  species  included  under  the  old  name  of  "Bacterium 
termo."  While  in  small  doses  and  under  ordinary  conditions  it  is  harmless,  at  times  and  in  large  doses  it  may  be  pathogenic. 

REFERENCES.    Lafar  194-199. 


MORPHOLOGICAL  CHARACTERS  . 

i 

~2 

V  3 

t" 

Incubation 
temp.  («C.) 

SKETCHES. 

i.  Form: 

b.  Agar  

c.  Gelatin  

d  Other  media  . 

2   Size. 

rf.  Special  stains  

5.  Motility  :  

a.  Character  of  movement  

b.  Flagella  stain  

6.  Spores  

7.  Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as : 

desiccation,  light,  disinfectants,  etc: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (r)  open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours . 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours ,48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


.  ..(2)  closed  arm: 

per  cent.,  72  hours per  cent., hours. 

...(5)    gas  formula,    H  :  CO,  :  :  : 


per  cent. 


,  to  ammonia. 


.  days 
.  days  . 


diastatic 


10.  Characteristic  odor. 

11,  Pathogenesis 


[72] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.C'C) 

24  HOURS 

48.   ..    .  HOURS 

0             Dws 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

• 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

(4) 
Agar 

Streak. 

x._'/ 
A 

A 

v         / 

A 

(5) 
Potato. 

Bouillon. 

(7) 

- 

Special 

Media. 

[73] 


Name  of  organism 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 

Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

t 

b    Loeffler's  methylen-blue  

5    Motility:          

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature: . 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites » 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours   ,48  hours 

9.  Enzyme  production:  proteolytic 


(2)     closed  arm : 

.per  cent.,  72  hours per  cent., 

(5)    gas  formula,  H  :  CO«  :  : 


.  hou  rs per  cent. 


,  to  ammonia 


.days, 
.days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[74] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  ("Q 


24 HOURS. 


48 HOURS. 


6 DAYS. 


SKETCHES. 


[75] 


A 


A 


Name  of  organism 
Source,  habitat,  etc.  . . 


MORPHOLOGICAL  CHARACTERS. 

en 
u 

«- 
n-.  s 
o~ 

85 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

• 

c   Gelatin                                                                   .                                                                               .... 

d   Other  media                 .                                                           ...                    .             

2   Size                                                 .                          ..            

X 

5.  Motility.                                         .... 

b.  Flagella  stain  .                    ... 

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.    Relation  to  free  oxygen: 

5.    Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 
4.    Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus   milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours  ,48  hours 

fecal  odor;  24  hours ,  48  hours 

9.  Enzyme  production  :  proteolytic 


....(2)    closed  arm:    

percent.,  72  hours per  cent hours  . 

..(5)    gas  formula,    H  :  CO2  :  : 


per  cent. 


,  to  ammonia . 


.  days, 
days. 


.  diastatic... 


10.  Characteristic  odor. 

11.  Hathogenesis 


[70] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium, 
Incubation 

24  ...   .  HOURS. 

48  ...  HOUKS. 

6            DAYS 

SKETCHES. 

Temp.  («C) 

(1) 

Gelatin 

plate: 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(2) 

Agar 

plate: 

(a)  Surface 

Colonies. 

(b)  Deep 

t 

Colonies. 

(3) 

. 

Gelatin 

Stab. 

- 

^ 

o 

(4) 
Agar 

A 

/\ 

Streak. 

i 

'N  ,X 

^ 

(5) 

A 

A 

A 

Potato. 

y 

& 

(6) 
Bouillon. 

(7) 
Special 

Media. 

[77] 


Name  of  organism  . . 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

i 

"°2 
£ 

Incubation 
temp.  (oC.) 

SKETCHES. 

i.  Form: 

c   Gelatin 

2  Size                 :  

5.  Motility                                  .             

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: , 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


...(2)  closed  arm: 

.per  cent.,  72  hours per  cent., 

•  ••(5)    gas  formula,   H  :  COs  :  : 


hours., 


percent. 


to  ammonia., 


days, 
days. 


.diastatic. 


10.    Characteristic  odor. 
u.    Pathogenesis 


[78] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 

24            HOURS 

48  HOURS. 

6  DAYS. 

SKET 

01 

ES 

Incubation 
Temp.  CC) 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

' 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

l> 

u 

(4) 

Agar 
Streak. 

•    •     ' 

A 

A 
/•  \ 

1         \ 

y 

V                 / 

(5) 
Potato. 

A 

A 

(6) 

.  '• 

Bouillon. 

^^ 

^^ 

(7) 

Special 

Media. 

- 

[79] 


CHAPTER  V. 

BACTERIOLOGICAL  ANALYSIS. 


EXERCISE  LXV.    COMPARATIVE  ANALYSIS  OF  AIR  (Koch's  Hethod). 

a.  Plate  three  tubes  of  gelatin  and  expose  by  removing  lid  for  20  minutes  in 
the  following  places:   1.  Laboratory,  2.  Cellar,  3.  Out  of  doors. 

b.  Replace  the  lids  and  keep  plates  at  22°  C.  for  several  days. 

c.  Count  the  colonies;   if  the  number  of  colonies  is  greater  than  100,  use  the 
counting  plate  figured  in  Plate  I.  A.  and  count  a  portion  and  estimate  the  whole  number. 

d.  Calculate  the  area  of  the  Petri  dish  by  multiplying  the  square  of  the  diameter 

by  0.785. 

e.  Express  the  results  in  terms  of  the  number  of  organisms  which  fall  per 
square  foot  per  minute. 

This  method  enables  one  to  make  a  rough  comparison  of  the  number  of  organisms 
occurring  in  the  localities  examined,  but  to  determine  the  number  per  volume  the  fol- 
lowing method  must  be  employed. 

REFERENCES.  H.  390. 

EXERCISE  LXVI.     QUANTITATIVE  DETERMINATION  OF  NUHBER  OF  BACTERIA  IN  AIR 
(Petri-Sedgwick  Method). 

GENERAL  DIRECTIONS. 

a.  A  piece  of  glass  tubing  6  mm.  (fin.) 
in  diameter  by  15  cm.  (6  in.)  long  is  drawn  out 
at  one  end  in  a  gas  flame  and  sealed. 

b.  Fill  this   tube   about  one-third  full 
with   granulated  sugar,  insert  a  cotton  plug  next  <x.- 
to  the  sugar  and  one  at  the   end   of  the  tube 
(Fig.13). 

c.  Sterilize  in  the  hot  air  sterilizer  for  "• 
1    and  Y-2.   hours   at  130°  C.  (sugar  melts  at  a 
higher  temperature).  V>. 

d.  Fasten  the  tube,  pointed  end  up,  in 
a  clamp,  remove  the  first  cotton  plug  and  con- 
nect with  an  aspirator.     (Fig.  14). 

FIG.  u.    Aspirator  for  e.  Break  off  the  pointed  end  of  the  tube 

filtering  air.  an(j  <jraw  a   measured  quantity  of  air  through  FIG.  is.   Apparatus  for  m- 

,-,  tering  air  through    sugar. 

the  sugar.  A  ready  for  sterilization. 

SPECIAL  DIRECTIONS.  B.  P°int  broken  off  and  at- 

TTvij.        rrv  TJ.  A     •  tached  to  aspirator. 

a.  Filter  50  liters  of  air. 

b.  Dissolve  sugar  in  10  cc.  of  sterile  water  and  make  plates,  using  1  cc.  of  the 
mixture. 

c.  Incubate,  count  colonies  as  above  and  estimate  the  number  of  organisms  per 
liter  of  air. 

REFERENCES.     A.  551;  H.  393;  L.  &  K.  392;  McF.  164;  N.  449;  S.  541. 


82 


General  Bacteriology. 


EXERCISE  LXVII.     RELATION  OF  BACTERIA  IN  THE  AIR  TO  DUST  PARTICLES. 

a.  Pour  a  tube  of  gelatin  into  a  Petri  dish  and  solidify. 

b.  Remove  the  lid  and  shake  a  dust-brush  or  cloth  over  it. 

c.  18-24  hours  later,  examine  under  low  power  of  microscope  to  determine  the  rela- 
tion of  the  developing  colonies  to  the  dust  particles. 

EXERCISE  LXVIII.     ESTIMATION  OF  NUMBER  OF  BACTERIA  IN  SOIL. 

a.  With  a  sterile  knife  collect  a  sample  of  soil  in  a  sterile  test-tube 
or  Petri  dish.     Samples  at  various  depths  can  be  secured  by  means  of  an 
earth  borer.     (Fig.  15). 

b.  Weigh  out  1  gram  and  dilute  1000  times  with  sterile  water. 

c.  Make  three  gelatin  plate  cultures  using  1  cc.,  £  cc.  and  rV  cc.    of 
this  suspension.     Incubate. 

d.  Count  the  colonies  as   they   develop   and  estimate  the  number   of 
bacteria  per  gram  of  soil. 

e.  Many   of  the   bacteria   of  the  soil  are  anaerobic  and  can  only  be 
grown  in  the  absence  of  free  oxygen.  See  Part  II.  Chap.  VII.  for  methods  of 
cultivation. 

REFERENCES.     A.  556;   H.  394;  L.  &  K.  389;   McF.  174;   N.  444;  S. 
567. 

Fig.  15.  Fraenkel's 
Soil  Borer. 

EXERCISE  LXIX.     WATER  ANALYSIS. 

QUANTITATIVE   ANALYSIS. 

a.  Collect  a  sample  of  water  in  a  sterile  test-tube  or  bottle.     Fig. 
16  shows  a  form  of  apparatus  used  in  taking  samples  of  water  at  vari- 
ous depths. 

b.  Make  two  gelatin  plates  using  ^  cc.   and  TV  cc.   of  the  water. 

c.  Count   the   colonies  as  they  appear,  and  estimate  the  number 
per  cc. 

d.  Make  agar  plates    and  compare  results  with  those    obtained 
above. 

e.  Analyze   a  surface   water    (lake  or  river) ,  a  deep  well  and   a 
spring  water. 

QUALITATIVE   ANALYSIS. 

a.  Detection  of  putrefactive  organisms.     Examine   gelatin  plates, 
made  above  and  (1)  determine  number  of  liquefying  organisms  per  cc. 
(2)  search  for  the  presence  of  proteus  forms.    (B.  vulgans.) 

b.  Detection  of  Faecal  Bacteria. 

1)  Inoculate  a  fermentation-tube  containing  glucose  bouillon  (1%)    with  1  cc. 
of  water. 

2)  Make  litmus  lactose  agar  plate  using  1  cc.  water. 

3)  Incubate  both  at  38°  C. 

4)  Compare  growth  obtained  with  that  of  B.  coli. 

REFERENCES.  A.  526;  H.  373;  L.  &  K.  396;  McF.  169;  M.  &  R.  79;  N.  422; 
P.  245;  S-  553.  For  the  determination  of  the  various  species  present  see  Frankland's 
Micro-organisms  of  Water;  Fuller:  Report  Am.  Public  Health  Assoc.,  1899,  580. 


---c. 


FIG.  18.    Russell's 
Water  Sampler. 


84  General  Bacteriology. 

EXERCISE  LXX.    QUANTITATIVE  ANALYSIS  OF  niLK. 

a.  Obtain  a  sample  of  milk  in  a  sterile  vessel. 

b.  Dilute  milk  1000  times  with  sterile  water. 

c.  Make  plates  as  under  soil  (LXVIII). 

d.  Count  colonies  and  estimate  number  of  bacteria  per  cc. 

EXERCISE  LXXI.     EFFICIENCY  OF  PASTEURIZATION. 

a.  Place  same  milk  as  used  in  previous  experiment  in  the  bottles  of  a  pasteurizing 
apparatus,  such  as  Freeman's,  and  pasteurize  as  per  printed  directions,  or  place  the  milk 
in  ordinary  milk  bottles  or  fruit  jars,  filling 

to  a  uniform  level;  these  are  then  to  be 
placed  in  a  flat  bottomed  pail  which  is  to  be 
filled  with  water  and  heated  to  71°  C.(160°  F. ) , 
remove  source  of  heat,  cover  and  allow  to 
stand  30  minutes.  Remove  bottles  and  cool 
as  quickly  as  possible  without  danger  to  glass. 

b.  Determine    bacterial  content    of    pas- 
teurized product  by  making  plates.     A  dilu- 
tion of  100   will  probably  be  sufficient.     Ex- 
press results  so  as  to  indicate  per  cent,  of  or- 
ganisms destroyed  by  the  process.     Compare 
the  keeping  qualities  of  the  pasteurized  pro- 
duct with  that  of  the  raw  milk  by   keeping 
samples    of    both   under  similar  conditions, 
e.  g.  in  locker  or  ice  chest,  making  frequent 
observations. 

Pasteurized  milk  should  not  have  a  permanently  cooked  taste. 

REFERENCES.     Bull.  Wis.  Exp.  Station  No.  44.     Russell,  Outlines  of  Dairy  Bacte- 
riology, 95  (4th  Edit.). 


FIG.  17.  A  home-made  pasteurizer  (Russell.) 


EXERCISE  LXXII.    TESTING  ANTISEPTIC  ACTION  OF  CHEMICALS. 

GENERAL  DIRECTIONS. 

o.  Fill  a  number  of  test-tubes  with  a  measured  quantity  of  agar  (5  cc.). 

b.  Add  to  the  agar  varying  but  measured  amounts  of  the  substance  to  be  tested.    If 
the  antiseptic  is  not  volatile,  or  affected  by  heat,  sterilize. 

c.  Inoculate  the  tubes  thus  prepared,  together  with  a  control,  with  B.  coli  and  make 
rolls. 

d.  Keep  these  cultures  under  observation  in  the  28°  C.  incubator- 

e.  If  no  growth  appears  within  96  hours  repeat  the  experiment,  using  smaller  amounts 
of  the  antiseptic.     In  this  way  determine  the  amount  of  chemical  (in  %)  which  just  pre- 
vents growth. 

SPECIAL  DIRECTIONS.     Test  in  this  way  carbolic  acid  (5  %),  alcohol  (95  %). 
REFERENCES.    A.  566;  H.  411;   N.  527;  S,  156. 


86  General  Bacteriology. 

EXERCISE  LXXIII.    TESTING  DISINFECTING  ACTION  OF  CHEMICALS. 

SUSPENSION   METHOD. 

a.  Make  a  culture  of  the  organism  to  be  studied  in  tubes  of  bouillon  containing  5  cc. 

b.  Incubate  at  38°  C.  for  24  hours. 

c.  Add  to  this  an  equal  amount  (5  cc.)  of  the  disinfectant  to  be  tested,  of  double  the 
required  strength. 

d.  At  the  end  of  5,  10,  20,  40,  and  60  minutes  make  agar  rolls,  using  two  or  three 
loopfuls  of  the  mixture  for  each  roll. 

e.  In  this  way  determine  the  time  of  exposure  necessary  to  kill  the  organism  used. 
/.  Test  in  this  way  the  value  of  corrosive  sublimate  (1: 1000)  and  Lysol  (5%),  using 

B.  coli. 

COVER -GLASS   METHOD. 

a.  Make  a  bouillon  culture  of  the  organism  to  be  studied  and  incubate  at  38°  C.for 
24  hours. 

b.  By  means  of  a  burette,  pipette,  or  loop,  place  the  same  sized  drop  on  each  of 
several  sterile  cover- glasses  and  dry  as  directed  in  the  experiment  on  desiccation  (XLIII). 

c-  When  the  cover-glasses  are  dry,  they  are  to  be  immersed  in  the  disinfectant  for 
the  desired  time,  then  removed  and  transferred  to  tubes  of  melted  agar  which  are  then 
made  into  rolls. 

d.  Test  by  this  method  carbolic  acid  (5%),  alcohol  (95%)  and  formaldehyde  (10%), 
using  B.  coli. 

REFERENCES.     A.  558;  N.  518;  P.  152;  S.  158. 


PA  RT    I  I. 

MEDICAL  BACTERIOLOGY. 


PART  II.— MEDICAL  BACTERIOLOGY. 
CHAPTER  VI. 

PATHOGENIC  AEROBES. 


EXERCISE  LXXIV.     PREPARATION  OF  CULTURE  MEDIA. 

The  following  media  will  be  necessary  for  the  work  outlined  in  the  following  chap- 
ters.    This  is  exclusive  of  a  few  special  media  which  are  described  under  special  heads 
and  are  to  be  performed  as  a  part  of  the  exercise  in  which  they  are  used. 
100  tubes  of  agar. 

12  tubes  of  glucose  agar. 
100  tubes  of  gelatin. 

12  tubes  of  glucose  gelatin. 

30  tubes  of  bouillon. 

30  fermentation  tubes  of  glucose  bouillon. 

30  tubes  of  potato. 

30  tubes  of  milk. 

30  tubes  of  glucose  free  broth  or  Dunham's  solution. 

30  water  blanks. 

30  tubes  of  blood  serum : 

a.  Collection  of  the  blood.  Sterilize  Mason  fruit  jars,  by  successive  washings  in 
corrosive  sublimate,  distilled  water,  alcohol  and  ether  (or  sterile  Erlenmeyer  flasks  may 
be  used) .  These  are  to  be  carried  to  the  slaughter  house  and  the  blood  from  a  beef  caught 
directly  into  them .  They  are  then  allowed  to  stand  undisturbed  for  15-30  minutes,  or 
until  the  clot  has  firmly  attached  itself  to  the  sides  of  the  vessel,  when  they  may  be 
removed  to  the  laboratory. 

6.  Separation  of  the  serum  from  the  blood  clot.  The  clot  is  separated  from  the 
sides  of  the  vessel  by  means  of  a  sterile  knife  or  glass  rod,  and  the  vessel  placed  in  the 
ice  chest.  After  standing  48  hours  the  clot  will  have  shrunken  away  from  the  walls  of 
the  vessel  leaving  the  clear  serum  on  the  top  and  at  the  sides.  This  can  now  be  pipetted  or 
siphoned  off.  If  the  serum  contains  a  large  number  of  red  blood  corpuscles  it  can  be 
placed  in  rather  tall  cylinders  (graduates)  and  allowed  to  stand  24  hours  longer,  when 
the  clear  straw  -colored  serum  can  be  readily  separated.  This  may  be  preserved  for  a 
long  time  by  the  addition  of  i  %  chloroform  and  kept  in  a  tightly  corked  bottle  in  a 
cool  place. 

c.  Loeffler's  mixture.     This  consists  of  3  parts  of  blood  serum  and  1  part  of  glu- 
cose bouillon  ( 1  % ) . 

d.  Sterilization.     Fill  sterile  test-tubes  (about  3  cm.  deep)  with  the  serum  and  ster- 
ilize either: 


Medical  Bacteriology.  89 

(1)  By  heating  to  60-65°  C.  for  1  hour  on  5  successive  days,  and  finally  plac- 
ing the  tubes  in  a  sloped  position  in  inspissator  (or  sloping  tray  in  a  high  temperature  in- 
cubator or  steamer)  and  heated  above  the  coagulating  point  of  the  serum  (70°  C.)  In  this 
method  the  clear  serum  is  used  and  not  Loeffler's  mixture  and  a  transparent  medium  ob- 
tained.    This  method  is  not  usually  employed,  but  the  following: 

(2)  Loeffler's  mixture  is  used  and  the  tubes  are  immediately  placed  in  a  sloping 
position  in  an  inspissator,  or  steamer  and  heated  up  to  95°  C.  for  1  hour  on  three  con- 
secutive days.     If  a  higher  temperature  is  employed  bubbles  are  formed  which  rupture 
the  surface  of  the  medium  in  their  escape.    When  sterile  the  tubes  should  be  sealed  with 
paraffin  or  otherwise. 

REFERENCES.     A.  106;  H.  45;  L.  &  K.  83;   M.  &  R.  50;   M.  &  W.  81;   McF.  131; 
N.  463;  P.  219;  S.  37  &  35. 


EXERCISE  LXXV.      STREPTOCOCCUS  PYOCJENES  Rosenbach. 

First  described  by  Rosenbach  in  1884.     It  is  found  in  abscesses,  pyemia,  puerperal  fever,  and  erysipelas.     It  is  frequently  present  in 
mixed  or  secondary  infections  and  occurs  in  the  mouth  and  sputum  and  on  the  mucous  membranes  of  the  nose,  urethra,  vagina,  etc. 

REFERENCES.     Rosenbach:  Mikroorganismen  bei  den  Wundinfectionskraukheiten  des  Menschen,  1884.      A.  268;  H.  133;  L.  &  K.  117; 
M.  &  R.  168;  M.  &  W.  124;  McF.  190;  P.  476;  S.  274. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 

Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

a.  Aqueous  gentian-violet  

5    Motility:     

a   Character  of  movement  

b   Flagella  stain  

" 

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: . 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

t.  Fermentation  tube,  growth  in:      (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours ,48  hours 

fecal  odor;  24  hours  ,48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  72  hours per  cent., hours.. 

......(5)    gas  formula,  H  :  CO«  :  : 


..  per  cent. 


,  to  ammonia 


.days, 
.days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[90] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 
Incubation 

Temp.  ("C 

24           HOURS 

48            HOURS 

0             DAYS 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

• 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 

Colonies. 

(b)  Deep 
Colonies. 

(3) 

• 

Gelatin 

Stab. 

A 

A 

/      \ 

'          \ 

(4) 

Agar 
Streak. 

v           . 
A 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

^ 

(7) 

Special 

Media. 

^_^ 

^^ 

[91] 


EXERCISE  LXXVI.    MICROCOCCUS  PYOQENES  (Rosenbach)  Mig. 

STAPHYLOCOCOUS  PYOQENES  ALBUS;  STAPHYLOCOOOUS  EPIDERMIDIS  ALBUS. 

First  described  by  Rosenbach,  in  1884.     One  of  the  common  organisms  found  in  pus.    Occurs  on  the  skin,  in  sputum,  air,  water,  dust 
and  soil. 

REFERENCES.     Rosenbach:  Mikroorganismen  bei  dem  Wundinfectionskrankheiten  des  Menschen.     1884.     McF.  184;  P.  470;  S.  272. 


MORPHOLOGICAL  CHARACTERS. 

<r> 

m 

h 

«-•  3 

3£ 

4>  ~ 

So 
< 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

2.  Size          

5.  Motility.               .                                 

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature  : 


t.    Relation  to  free  oxygen: 

5.    Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 
4.    Pigment  production: 


}.    Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus   milk 

7.  Reduction  of  nitrates:  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

g.    Enzyme  production  :  proteolytic 


—  (a)  closed  arm: 
per  cent.,  72  hours 
•  •(5)  gas  formula,  H 


per  cent  ................  hours 


per  cent. 


,  to  ammonia. 


.  days, 
days. 


.  diastatic — 


10.  Characteristic  odor. 

11.  Pathogenesis 


[92] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  (°C) 


24 HOURS. 


48...      .  HOURS. 


0..       .  DAYS. 


SKETCHES. 


,-"• 


/\ 


A. 


[93] 


EXERCISE  LXXVII.     MICROCOCCUS  HELTINESIS  Bruce. 

This  organism  is  the  cause  of  Malta  (ever  and  is  found  especially  in  the  spleen  of  the  diseased. 

REFERENCES.     Bruce:    Ann.  de  1'  Inst.  Pasteur,  1899,  8;  239.     Durham:    Jour.  Path,  and  Bact.,  1898,  5;  377.     H.  301 ;  M.  &  E.  449. 


MORPHOLOGICAL  CHARACTERS. 

vi 
H 

'oB 

8? 
<° 

Incubation 
temp.  <<>C.) 

SKETCHES. 

i.  Form: 

e.  Gelatin    ...             

2   Size 

d  Special  stains 

5    Motility 

b   Flagella  stain                                                                                                                                                  .  .             

• 

PHYSIOLOGICAL  CHARACTERS. 


r.    Relation  to  temperature  :. . 


2.  Relation  to  free  oxygen: , 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm; 

6.  Acid  or  alkali  production,  litmus  milk 

y.    Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours , 

9.  Enzyme  production :  proteolytic 


...(2)  closed  arm: 

.per  cent.,  72  hours per  cent., 

...(5)    gasformula,   H  :  CO*  :  : 


hours., 


per  cent. 


to  ammonia.. 


days 

days 


.diastatic. 


10.  Characteristic  odor.. 

11.  Pathogenesis 


[94] 


CULTURE  CHARACTERS. 


Kc:u  lion 
of  Medium. 
Incubation 
Temp.  («C) 

''4           HOURS 

48  HOURS. 

0  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

. 

A 

A 

/  \ 

(4) 
Agar 

Streak. 

• 

'v  X 

A 

A 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

- 

Special 

Media. 

^ 

[95] 


EXERCISE  LXXVIII.    MICROCOCCUS  AUREUS  (Rosenbach)  Mig. 

STAPHYLOCOCCUS  PYOGENES  AURKUS  ;  GOLDEN  PUS  ooocus. 
First  described  in  1884  by  Eosenbach.     It  is  the  most  common  organism  in  pus.— 80^. 

REFERENCES.      Eosenbach:  Mikroorganismen  bei  dem  Wundinfektionskrankheiten  des  Mensohen.  A.  260;  H.  130;  L.  &  K.  115;  M.  & 
E.  160;  M.  &  W.  121  ;  McF.  184;  P.  401 ;  S.  265. 


MORPHOLOGICAL  CHARACTERS. 

• 

E 

"32 

V  3 
1° 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin 

2   Size 

b.  Loeffler's  methylen-blue 

b.  Flagella  stain  

6,  Spores  

7.  Special  characters,  such  as:  ..  . 

deposits,  vacuoles  

pleimorphic  and  involution 

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc: 

4.  Pigment  production:. 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm : 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours ,48  hours 

9.  Enzyme  production  :  proteolytic 


. .  ..(2)  closed  arm: 

per  cent.,  72  hours per  cent hours 

...(5)    gas  formula,    H  :  CO,  :  :  : 


per  cent. 


,  to  ammonia. 


.  days 
days  . 


diastatic 


10.  Characteristic  odor 

11.  Pathogenesis 


[96] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 
Incubation 
Temp.  («C) 

24           HOURS 

48  .   .      HOURS 

6  .   .      DAYS 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies 

(b)  Deep 
Colonies 

(2) 

Agar 
plate  : 

(a)  Surface 

Colonies. 

(b)  Deep 
Colonies. 

t 

(3) 

Gelatin 

Stab. 

A 

A 

'         \ 

(4) 

Agar 
Streak. 

X 

A 

/  \ 

k        y 

A 

(5) 
Potato. 

\ 

(6) 

Bouillon. 

•      - 

- 

V 

(7) 

Special 

Media. 

V   / 

[97] 


EXERCISE  LXX1X.     MICROCOCCUS  GONORRHOEA  (Neisser)  Fluegge 

GONOCOCOUS  ;  DlPLOCOCCUS  OF  GONORRHOEA. 

First  described  in  1879  by  Neisser.  It  is  constantly  found  in  gonorrhoeal  discharges  and  may  produce  disease  on  any  mucous  mem- 
brane; urethra,  bladder,  rectum,  conjunctiva  (causing  ophthalmia  neonatorum),  and  even  cause  arthritis  (gonorrhoea!  rheumatism), 
endocarditis,  salpingitis  and  general  septicaemia. 


L.  &  K.  311  ;M.  &  R.  189;  M.  &  W.  130;  McF.  201  ;  P.  522;  S.  288. 

MORPHOLOGICAL  CHARACTERS. 

Age  of 
Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

2   Size 

b.  Loeffler's  methylen-blue  

c.  Gram's  stain  

d.  Special  stains  

5.  Motility:  

a.  Character  of  movement  

b.  Flagella  stain  ;  

6.  Spores  

7.    Special  characters,  such  as:  ..".  .  

deposits,  vacuoles  :  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 

:.    Relation  to  temperature: 


a.    Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: . 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

A.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours  48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.  per  cent.,  72  hou  rs per  cent., hou  rs . 

(5)    gas  formula,  H  :  COi  :  :  : 


per  cent. 


,  to  ammonia 


.days, 
.days. 


.diastatic. 


10.    Characteristic  odor, 
n.    Pathogenesis 


[98] 


Medical  Bacteriology.  99 

The  Micrococcus  gonorrhoeae  does  not  grow  on  the  ordinary  artificial  media  but  may 
be  cultivated  on  the  following: 

a.  Blood  agar.  Blood  drawn  from  the  finger,  under  aseptic  precautions,  in  a  cap- 
illary pipette  is  placed  on  the  surface  of  agar  either  in  tube  or  Petri  dish.  This  blood 
is  then  inoculated  with  the  material  containing  the  organism  (pus  or  pure  culture)  and 
smeared  over  the  surface  of  the  agar  either  with  the  loop  or  better  by  means  of  a  sterile 
camel's  hair  brush. 

6.  Wertheim's  method.  Human  blood-serum  (from  placenta  or  pleuritic  or  other 
effusion  may  be  used)  in  a  fluid  and  sterile  condition  is  placed  in  two  or  three  test-tubes. 
These  are  heated  to  40°  C.  and  inoculated  with  the  material  containing  the  organism, 
making  dilutions  from  one  to  another  if  necessary.  To  each  tube  is  then  added  an 
equal  quantity  of  nutrient  (ordinary  or  2%)  agar  thoroughly  liquefied  and  cooled  to 
40°  C.  The  two  are  then  thoroughly  mixed  and  quickly  poured  into  Petri  dishes  and 
placed  in  the  incubator  at  38°  C.  Colonies  appear  in  24  hours. 

c.  Rabbit  blood-serum  may  be  used  either  in  a  fluid  or  solid  condition. 


EXERCISE  LXXX.    MICROCOCCUS  INTRACELLULARIS  (Weichselbaum)  Mig. 

DIPLOCOOCUS  OF  CEREBRO-SPINAL  MENINGITIS. 

First  described  in  1887  by  Weichselbaum.  It  is  found  in  the  meningeal  exudate  of  certain  cases  of  epidemic  cerebro-spinal  menin- 
gitis and  in  nasal  secretions  in  a  number  of  cases. 

REFERENCES.  Weichselbaum:  Fortschritte  der  Medicine,  1887;  Councilman :  Rept.  Mass.  State  B.  of  H.  1898;  A.  285;  H.  138;  M.  & 
R.  172;  M.  &  W.  135;  P.  516;  S.  310. 


MORPHOLOGICAL  CHARACTERS. 

• 

V 

3 

o- 

O  = 

BU 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

« 

c   Gelatin                               .     .  .'  

2   Size                                    

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


a.    Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  14  hours percent..  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus   milk 

~.    Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic  


....(21    closed  arm:    

percent.,  72  hours  per  cent hours per  cent. 

..(5)    gas  formula,    H  :  COZ  ::  : 


,  to  ammonia . 


.  days. 
.  days. 


.  diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[100] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  (°C) 

24            HOURS 

48            HOURS 

0             DAYS 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

• 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

/\ 

A 

(4) 

Agar 
Streak. 

(5) 
Potato. 

(6) 

Bouillon. 

• 

- 

- 

(7) 

Special 

Media. 

[101] 


EXERCISE  LXXXI.     SARCINA  TETRAQENA  (Qaffky)  Mig. 

MlCROCOCCTJS  TETRAGENUS. 

First  described  in  1883  by  Gaffky.     It  is  found  in  phthisical  cavities  and  sputum  and  it  occasionally  occurs  in   mixed  infections  as 
abscesses  connected  with  carious  teeth,  about  the  neck,  jaws,  and  middle  ear,  rarely  elsawhere. 

REFERENCES.     Gaffky:  Langenbeck's  Archev,  1883,  28:500.     A.  309;  H.  130;  M.  &  R.  171 ;  M.  &  W.  133;McF.  443 ;  P.  472 ;  S.  314. 


MORPHOLOGICAL  CHARACTERS. 

8 

"33 
$ 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatlii 

z   Size 

a.  Aqueous  gentian-violet  

* 

b.  Loeffler's  methylen-blue  

c.  Gram's  stain  

5.  Motility:  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.; 

4.  Pigment  production: 


5-    Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  ol  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


... (2)  closed  arm: 

.percent.,  72  hours percent.. hours per  cent. 

•  ••(5)    gas  formula,   H  :  COj  :  :  : 


to  ammonia., 


days. 
days. 


.diastatic. 


10.  Characteristic  odor., 

1 1 .  Pathogenesis 


[102] 


CULTURE  CHARACTERS. 


(1) 


(2) 


(3) 


Reaction 
of  Medium 
Incubation 
Temp.  ("C 


(4) 


(5) 


Potato. 


(6) 


Bouillon. 


Special 
Media. 


24. .     . .  HOURS. 


48  HOURS. 


(5 . .       .  DAYS. 


SKETCHES. 


A 

(\ 


[103] 


EXERCISE  LXXXII.     BACTERlUn  ANTHRACIS  (Koch)  nig. 

BACILLUS   OF   ANTHRAX. 

First  described  by  Robert  Koch  in  1876.     Found  in  the  blood  and  tissue  in  cases  of  anthrax  or  splenic  fever. 

REFERENCES.     Koch:  Cohn's  Beitraege  zur  Biologie  der  Pflanzen,  1870,  2;  277.      Chester:    Dept.  Delaware   Exp.  Station,  July,  1895. 
A.  448;     H.  151 ;  L.  &  K.  287;  M.  &  R.  295;  M.  &  W.  156;  McF.  356;  P.  547;  S.  328. 


MORPHOLOGICAL  CHARACTERS. 

i 

°s 

41  3 

*5 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin  . 

d  Other  media  . 

2   Size. 

_ 

d.  Special  stains  

5.  Motility  :  

b.  Flagella  stain  

6.  Spores  

7.  Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as : 

desiccation,  light,  disinfectants,  etc: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

ft.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production :  proteoly tic 


...(2)  closed  arm: 

per  cent.,  72  hours per  cent 

...(5)    gas  formula,    H  :  COj  :  : 


hours. 


per  cent. 


,  to  ammonia . 


.  days 
days  . 


.  diastatic 


10.  Characteristic  odor. . 

1 1 .  Pathogenesis 


[104] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium 
Incubation 

24           HOURS 

48  HOURS. 

6  DAYS. 

SKETCHES. 

Temp.  (°C 

(1) 

Gelatin 

plate  : 

(a)  Surface 

Colonies 

(b)  Deep 

Colonies 

(2) 

.  i 

Agar 

plate  : 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(3) 

Gelatin 

Stab. 

o 

^ 

(4) 
Agar 

- 

A 

/\ 

J          1 

Streak. 

i 
i 

•X«,    ^^ 

(5) 

A 

A 

/               4 

/     \ 

/                   1 

i      \ 

Potato. 

& 

(6) 
Bouillon. 

& 

sJ 

(7) 

Special 

Media. 

^ 

[105] 


EXERCISE  LXXXIII.    BACTERIUM  PNEUMON1AE  (Welchselbaum)  Mlg. 

PNEUMOCOCCUS  ;  DIPLOOOCOUS  OP  PNEUMONIA;  MICROOOCOOS  LANCEOLATTJS. 

First  described  by  Sternberg  in  1880.  Found  in  saliva  and  nasal  secretion  of  healthy  persons — about  20  per  cent.  Usually  present 
in  "rusty  sputum"  of  pneumonia. 

EEPEEENOES.  Weichselbaum :  Am.  Jour.  Med.  Sci.,  July,  1886:  Welch:  Johns  Hop.  Hosp.  Bulletin,  1892,  8;  125;  A.  303.  H.  273;  L. 
&  K.  118;  M.&  R.  204;  M.  &  W.  128;  McF.  345;  P.  498;  S.  298. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 
Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin 

2   Size         

in  growths  

a.  Aqueous  gentian-violet  S.  

b.  Loeffler's  methylen-blue  

d.  Special  stains  

5.  Motility:  - 

a.  Character  of  movement  

b.  Flagella  stain  

6.  Spores  

7.    Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.     Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.    Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:      (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  , 

7.  Reduction  of  nitrates;  to  nitrites  

8.  Indol  production;  24  hours ,48  hours 

fecal  odor;  24  hours  ,48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  72  hours percent., hours. 

(5)    gas  formula,  H  :  CO«  :  :  : 


per  cent. 


.  to  ammonia 


. days . 
. days . 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[100] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  (°C) 

24  HOURS 

48  HOURS 

6    .        DAYS 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

- 

Stab. 

A 

A 

l\ 

/\ 

A 

(4) 

Agar 
Streak. 

(5) 
Potato. 

(0) 
Bouillon. 

Special 

Media. 

^ 

^^ 

[107] 


EXERCISE  LXXXIV.    BACTERIUM  PNEUHONICUn  (Friedlander)  nig. 

FRIEDLANDER'S  BACILLUS. 

First  described  by  Friedlander  in  1882.     Found  frequently  in  normal  saliva,  lungs,   "rusty  sputum"  of  pneumonia,  and  has  been 
found  in  air  and  water. 

REFERENCES.     Friedlander:  Virchow's  Archiv,  32;  319;  H.  278;  L.  &  K.  119;  M.  &  R.  211;  McF.  353;  P.  458;  S.  296. 


MORPHOLOGICAL  CHARACTERS. 

en 

|B 

Incubation 
temp.  (°C.) 

SKETCHES. 

x.  Form: 

b.  Loeffler's  methylen-blue  

5    Motility 

7,  Special  characters,  such  as:  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  :. 


2.    Relation  to  free  oxygen: 

j.    Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: . 
4.    Pigment  production:.. 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent..  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic .-. 


....(2)    closed  arm:    

per  cent.,  72  hours per  cent 

..(5)    gas  formula.    H  :  COi  :  : 


—  hours... 


per  cent. 


,  to  ammonia. 


.  days. 
.  days. 


.  diastatic. 


10.    Characteristic  odor. 
n.    Pathogenesis 


[108] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 
Incubation 
Temp.  (°C; 

24  ...  HOUES. 

48  HOURS. 

C            DAYS 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

. 

(3) 

Gelatin 

Stab. 

A 

A 

!\ 

Agar 

Streak. 

'N  / 

A 

f  \ 

>•            X 

A 

(5) 
Potato. 

(6) 

Bouillon. 

^ 

- 

Special 

Media. 

^  ) 

[109] 


EXERCISE  LXXXV.     BACTERIUH  CUNICULICIDA  Koch. 

BACILLUS  OP  CHICKEN  CHOLERA;  BACILLUS  OF  SWINE  PLAGUE;  BACILLUS  SEPTICAEMIAS  HEMORRHAGICAE. 

First  described  by  Koch  in  1878.  Found  in  blood,  organs  and  excreta  of  chickens  suffering  with  fowl  cholera,  and  swine  suffering 
from  swine  plague. 

REFERENCES.  Koch:  Wundiiifektionskrankheiten.  Septikaemie  bei  Kaninchen,  1878;  Smith:  Report  on  Swine  Plague,  Bureau  of 
Animal  Industry,  U.  S.  Dept.  Agri.,  1891 ;  Smith  &  Moore:  Bull.  6,  B.  A.  I.,  1894;  H.  208;  McF.  409;  S.  408. 


MORPHOLOGICAL  CHARACTERS. 

tn 

s 

~^2 

w"a 

& 

Incubation 
temp.  (oC.) 

SKETCHES. 

i.  Form: 

* 

c   Gelatin                                      

2   Size                                       .... 

5    Motility                     

b    Flagella  stain                .  .  .".  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light ,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

A.  Fermentation  tube,  growth  in:    (t)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours ,  48  hours 

fecal  odor;  24  hours 48  hours 

9.  Knzyme  production  :  protcolytic 


...(2)  closed  arm: 

.percent.,  72  hours per  cent 

...(5)    K<TS  formula,   H  :  COj  :  : 


hours per  cent. 


to  ammonia.. 


days, 
days. 


.diastatic. 


TO.    Characteristic  odor. . 
ii.    Pathogenesis 


[110] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 
Incubation 
Temp.  (°C 


24 HOURS. 


48 HOURS. 


0 . . .      .  DAYS. 


SKETCHES. 


(1) 

Gelatin 
plate: 

(a)  Surface 
Colonies, 

(b)  Deep 
Colonies. 


(2) 

Agar 
plate : 

(a)  Surface 
Colonies 

(b)  Deep 
Colonies 


(3) 

Gelatin 
Stab. 


(4) 

Agar 
Streak. 


(5) 


Potato. 


(6) 


Bouillon. 


(7) 

Special 
Media. 


A 


A 

/\ 


A 

/\ 


A 


[ill] 


EXERCISE  LXXXVI.     BACTERIUn  RHUSIOPATHIAE  (Kitt)  nig. 

BACILLUS  OF  SWINE  ERYSIPELAS:  ROUGET. 
First  described  by  Loeffler  in  1882.     Found  in  blood,  internal  organs,  etc.,  of  swine  infected  with  the  disease. 


MORPHOLOGICAL  CHARACTERS. 

i 

•S2 

V  3 
1" 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

b.  Agar  

c.  Gelatin  

d.  Special  stains  

5.  Motility  :  

a.  Character  of  movement  . 

b,  Flagella  stain  

6.  Spores  

7.  Special  characters,  such  as:  ... 

deposits,  vacuoles  

pleomorphic  and  involution 

forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours ,48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


...  (2)  closed  arm: 

percent.,  72  hours percent., hours per  cent. 

•  ••(5)    gas  formula,    H  :  CO«  :  :  : 


,  to  ammonia . 


.  days 
days  . 


diastatic 


10.    Characteristic  odor. .. 
u.    Pathogenesis 


[112] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 

•>4           HOURS 

48           HOURS 

6             DAYS 

SKEI 

I  '1 

tES 

Incubation 
Temp.  (°C 

(1) 

Gelatin 

plate  : 

(a)  Surface 
-    Colonies 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 

Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

o 

u 

(4) 

Agar 
Streak. 

A 

A 

A 

^N  ,S 

v    / 

(5) 
Potato. 

• 

A 

t  \ 

A 

(6) 

Bouillon. 

^, 

^^ 

(7) 

Special 

Media. 

• 

^  J 

[113] 


EXERCISE  LXXXVII.     BACTERIUH  TUBERCULOSIS  (Koch)  dig. 

BACILLUS  OF  TUBERCULOSIS. 
First  described  by  Koch  in  1882.     Found  in  diseased  tissue  of  man  and  animals  and  phthisical  sputum. 

REFERENCES.     Koch:  Berlin.  Klin.   Wochenschr.,  1882,  15;  221;  Smith:    Jour.  Exp.  Med.,  1898,  3:  451;  A.  312;  H.  189;  L.  &  K.  251; 
M.  &  R.,  224;  M.  &  W.  148;  McF.  208;  P.  263;  S.  375. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 
Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                          

a.  Aqueous  gentian-violet  .'  

b.  Loeffler's  methylen-blue  

c.  Gram's  stain  

d  Special  stains  -  

5.  Motility:  

a    Character  of  movement  

b   Flagella  stain  

pleomorphic  and  involution  forms,  capsules,  etc  

•  PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature: . 


j.    Relation  to  tree  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:      (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

T.    Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  14  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  73  hours per  cent., hours... 

(5)    gas  formula,  H  :  COj  :  :  : 


percent. 


,  to  ammonia  . 


.days, 
.days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[114] 


Medical  Bacteriology.  115 

B.  tuberculosis  does  not  grow  upon  the  ordinary  artificial  media,  but  may  be  grown 
upon  blood  serum  [see  p.  89  (1)  ]  and  bouillon,  agar  and  potato  to  which  5%  of  glyc- 
erine has  been  added.  The  tubercle  bacterium  is  very  sensitive  to  temperature  varia- 
tions and  should  therefore  be  kept  at  a  temperature  varying  at  most  only  a  degree  or 
two  from  38°  C.  It  is  also  extremely  sensitive  towards  desiccation  and  for  this  reason  the 
cotton  plug  should  be  well  paraffined  or  replaced  by  a  cork  through  which  a  small  cotton 
plugged  glass  tube  passes  and  the  incubator  kept  saturated  with  moisture.  For  methods 
of  culture  and  isolation  see  Smith:  Jour.  Exp.  Med.,  1898,  3;  456. 


EXERCISE  LXXXVIH.     BACTERIUM  MALLEI  (Loeffler)  Mig. 

BACILLUS  OP  GLANDERS. 

First  described  by  Loeffler  in  1886.     Found  in  the  nodules,  ulcers,  discharges,  etc.,  of  glanders  or  farcy. 

REFERENCES.  Loeffler:   Arbeit,  aus  dem  Kais.   Gesundheitsamte,  1886,  1 ;  141 :  A.  339;  H.  217;  L.  &  K.  300;  M.  &  R.  268;  M.  &  W.  164; 
McF.  248;  P.  598  ;S.  39C. 


MORPHOLOGICAL  CHARACTERS. 

• 
v 

3 

o±: 

&3 

< 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                          

2   Size                                 

5    Motility                       

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: . 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

j.    Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


....(2)    closed  arm:    

per  cent.,  72  hours per  cent.,  

..(5)    gas  formula,    H  :  COZ  :  : 


hours per  cent. 


,  to  ammonia. 


.  days, 
days. 


.  diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[110] 


CULTURE  CHARACTERS. 


Reaction 
ol  Medium. 
Incubation 
Temp.  (°C) 

24           HOURS. 

48  .  .     HOURS 

C  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

B 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

A 

(4) 

Agar 
Streak. 

'    • 

'v  / 

A 

i\ 

v             / 

A 

(5) 
Potato. 

- 

^J 

v    J 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

Media. 

V  ) 

[117] 


EXERCISE  LXXXIX.     BACTERIUM  DIPHTHERIAS  (Loeffler)  Mig. 

BACILLUS  OF  DIPHTHERIA.;  KLEBS-LOEFFLER  BACILLUS. 

First  described  in  1883  by  Klebs.  First  cultivated  in  1884  by  Loeffler.  Found  in  the  false  membrane  in  cases  of  diphtheria  and  in 
small  numbers  in  spleen,  liver,  etc. ;  occasionally  in  healthy  throats. 

REFERENCES.  Klebs:  Verhandl.  d.  Kongressfuer  innere  Medizin,  1883,  II.  Loeffler:  Mitth.  aus  dem  Kais.  Gesundheitsamte,  1884, 
2;  421.  A.  349;  H.  162;  L.  &  K.  207;  M.  &  R.  !i53;  M.  &  W.  137;  McF.  284;  P.  329;  S.  356. 


MORPHOLOGICAL  CHARACTERS. 

• 

V 

"ol 
'"'3 

#3 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                               .             .                          .             .          ... 

d  Other  media              .                    

2   Size                                      .                                                                  -             

* 

5^  Motility 

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light ,  disinfectants,  etc.:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

y.    Reduction  of  nitrates:  to  nitrites 

8.  Indol  production:  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Knzyme  production  :  proteolytic 


...(2)  closed  arm: 

.percent.,  72  hours percent hours. 

...(i)    gas  formula,  H  :CO,  :  :  : 


per  cent. 


to  ammonia.. 


days. 


days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[118] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 
Incubation 
Temp.  (°C) 

24           HOURS 

48  HOURS. 

6  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

A 

'         \ 

A 

(4) 

Agar 
Streak. 

(5) 
Potato. 

(6) 

• 

Bouillon. 

^ 

- 

(7) 

Special 

Media. 

• 

[119] 


EXERCISE  XC.    BACTERIUCl  INFLUENZAE  (R.  Pfeiffer)  Lehm.  &  Neum. 

BACILLUS  OF  INFLUENZA;  LA  GRIPPE. 

First  described  in  1892  by  R.  Pfeiffer.     Found  in  the  sputum  and  nasal  secretions  of  the  diseased. 
REFERENCES.     Pfeiffer:  Z.  f.  H.   1893,  13;    357;  A.  334;  H.  280;  L.  &  K.  281  ;M.  &  R.  431;  M.  &  W.  162;  McF.  440;  P.  320;  S.  370. 


MORPHOLOGICAL  CHARACTERS. 

| 

•32 

4)   3 

& 

Incubation 
temp.  (°C.) 

SKETCHES. 

x.  Form: 

c   Gelatin  . 

d.  Other  media  . 

2.  Size. 

5.  Motility: 

a.  Character^  movement  

If.  Flagella  stain  

6.  Spores  r  

7.  Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


i.    Relation  to  free  oxygen:, 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc:. . . 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

A.  Fermentation  tube,  growth  in:    (j)  open  arm: (2)  closed  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours percent.,  72  hours percent hours per  cent. 

(4)  reaction  in  open  arm: (5)    gas  formula,    H  :  COj  :  :  : 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites ,  to  ammonia 

8.  Indol  production;  24  hours ,48  hours days 

fecal  odor;  24  hou rs 48  hou rs days 

g.    Enzyme  production :  proteolytic diastatic  


10.  Characteristic  odor. 

11.  Pathogenesis 


[120] 


Medical  Bacteriology.  121 

B.  influenzae  does  not  grow  on  the  ordinary  artificial  culture  media  but  may  be  cul- 
tivated on  agar  slopes  upon  the  surface  of  which  blood  has  been  smeared.  The  blood 
from  man,  rabbits,  guinea-pigs  and  frogs  can  be  used,  but  that  from  pigeons  is  best. 
The  blood  may  be  obtained  from  a  needle  prick  and  spread  over  the  medium  with  a  loop. 
The  skin  should  first  be  washed  with  alcohol  and  then  ether  and  the  first  drops  should 
not  be  used.  The  sterility  of  these  tubes  should  be  tested  by  placing  them  in  an  incu- 
bator for  24  hours  previous  to  inoculation. 


EXERCISE  XCI.     BACILLUS  TYPHOSUS  Oaffky. 

BACILLUS  OF  TYPHOID  FEVER;  EBERTH'S  BACILLUS. 

First  described  by  Eberth  in  1880,  first  cultivated  by  Gaffky,  1884.     It  is  found  in  the  faeces  and  urine  of  typhoid  patients. 

REFERENCES.     Eberth:  Virchow's  Archiv.  1880,  81;  58  and  1881,  83;  480.     Gaffky:  Mitth.  aus  dem  Kais.  Gesundheitsamte,   1884,  2; 
372;  A.  309;  H.  223;  L.  &  K.  16(i;  M.  &  R.  317;  M.  &  W.  141;  McF.  306;  P.  402;  S.  337. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 
Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

,- 

b   Loeffler's  methylen-blue  

5    Motility                        

7     Special  characters,  such  as:  

PHYSIOLOGICAL  CHARACTERS. 


i.     Relation  to  temperature:  . 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:    24  hours — per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates:  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  72  hours percent hours. 

(5)    gas  formula,  H  :  CO.  :  : 


per  cent. 


,  to  ammonia 


.days. 
. days . 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[122] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium. 
Incubation 

24      .     HOURS 

48  HOURS. 

6  DAYS. 

SKETCHES. 

Temp.  (UC) 

(1) 

Gelatin 

plate  : 

• 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(2) 

Agar 

plate  : 

(a)  Surface 

Colonies. 

•- 

(b)  Deep 

Colonies. 

(3) 

Gelatin 

Stab. 

^ 

^ 

(4) 
Agar 

A 

A 
/     \ 

f         \ 

Streak. 

i 
i 

^/ 

\Tx 

i 

i 

(5) 

A 

/  \ 

A 

Potato. 

« 

& 

^ 

(6) 
Bouillon. 

A 

^ 

w 

(7) 

Special 

Media. 

[123] 


EXERCISE  XCII.    BACILLUS  PESTIS  Kitasato  and  Yersin. 

BACILLUS  OF  BUBONIC  PLAGUE. 

Described  at  about  the  same  time  independently  by  Kitasato  and  Yersin  in  1894.  Found  in  the  buboes,  and  occasionally  in  the  faeces, 
urine  and  blood  and,  in  the  pneumonic  form,  in  the  blood. 

EEFERENCES.  Kitasato:  Lancet,  1894,  3;  428;  Yersin:  Ann.  de  1'  Inst.  Past.,  1894,  8;  663;  A.  393;  H.  359;  L.  &  K.  300;  M.  &  R.  437; 
McF.  483;  P.  606. 


MORPHOLOGICAL  CHARACTERS. 

V 

**-  s 

Incubation 
temp.  (°C.J 

SKETCHES. 

i.  Form: 

' 

c   Gelatin     ".  

d   Other  media  

2   Size         

5    Motility                                                                                               .                                                        .... 

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Figment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus   milk 

7 .  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteoly  tic 


....(2)    closed  arm:    

per  cent.,  72  hours percent., hours. 

..(5)    gas  formula,    H  :  CO2  :  :  : 


per  cent. 


,  to  ammonia. 


.  days, 
days. 


.  diastatic. 


10.  Characteristic  odor. 

11.  Hathogenesis 


[134] 


CULTURE  CHARACTERS. 


• 

Reaction 
of  Medium, 
Incubation 
Temp.  (°C) 

24            HOUKS 

48               HOURS 

G            DAYS 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(8) 

Gelatin 

Stab. 

A 

/\ 

(4) 
Agar 

Streak. 

i 

^  / 

A 
/    \ 

A 

(5) 

Potato. 

^^ 

^^ 

Bouillon. 

(7) 

Special 

Media. 

0 

[125] 


EXERCISE  XC1II.    BACILLUS  SUIPESTIFER  Kruse. 

BACILLUS  OF  HOG  CHOLERA. 

First  described  by  Klein,  1884,  first  cultivated  by  Salmon  and  Smith  in  1885.     Occurs  in  blood,  organs  and  intestinal  contents  of  hogs 
suffering  from  hog  cholera. 

REFERENCES.    Salmon  &  Smith :  Kept   Bureau  Anim.  Ind.,  1885-91;  H.  269;  McF.  413;  S.  413. 


MORPHOLOGICAL  CHARACTERS. 

• 
£ 
~°S 

<U*3 
fO 

Incubation 
temp.  (oC.) 

SKETCHES. 

i.  Form: 

c   Gelatin                        

5    Motility                                                                                                                                           -                          ... 

,  ••  \  

PHYSIOLOGICAL  CHARACTERS. 


x.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  iu:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates:  to  nitrites 

8.  Indol  production:  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


...(2)  closed  arm: 

.  per  cent.,  72  hours per  cent., 

...(5)    gas  formula,   H  :  CO8  :  : 


hours per  cent. 


to  ammonia.. 


days, 
days. 


.diastatic. 


10.  Characteristic  odor. .. 

11.  Pathogenesis 


[126] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 
Incubation 
Temp.  (»C) 

24               HOUKS 

48  HOURS. 

C    .  .      DAYS. 

SKETCHES. 

0) 

Gelatin 

. 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies 

(3) 

Gelatin 

Stab. 

A 

A 

A 

A 

A, 

(4) 

Agar 
Streak. 

* 

(5) 
Potato. 

I 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

Media. 

- 

[137] 


EXERCISE  XCIV.     BACILLUS  ICTEROIDES  Sanarelli. 

First  described  in  1807  by  Sanarelli,  and  claimed  by  him  to  be  the  cause  of  yellow  fever. 
REFERENCES.     H.  369;  M.  &  R.  453;  McF.  400;  P.  609. 


MORPHOLOGICAL  CHARACTERS. 

1 

'oH 
<^ 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

d  Other  media  .                                  .                    .             .                    . 

2   Size.                                                                                    

5.  Motility  :                                  .                            .                                                                                              ...... 

a.  Character  of  movement  

A.  Flagella  stain  

6,  Spores  

7.  Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as : 

desiccation,  light,  disinfectants,  etc:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

A.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours. 

(4)  reaction  in  open  arm : 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours ,48  hours 

9.  Enzyme  production  :  proteolytic 


.  ..(2)  closed  arm: 

per  cent.,  72  hours per  cent., 

-•-(5)    gas  formula,    H  :  COj  :  : 


hours... 


per  cent. 


,  to  ammonia. 


.  days 
.  days  . 


diastatic 


10.  Characteristic  odor. . 

11.  Pathogenesis 


[128] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.("C 

24            HOURS 

48  HOURS. 

c  DAYS 

SKETCHES. 

(1) 

Gelatin 

plate  : 

. 

(a)  Surface 
Colonies 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

, 

Stab. 

A 

*>  —  y 

A 

A 

A 

A 

(4) 

Agar 
Streak. 

(5) 
Potato. 

*& 

X         x' 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

Media. 

[129] 


EXERCISE  XCV.    PSEUDOMONAS  AERUQINOSA  (Schroeter)  nig. 

BACILLUS  PYOCYANEUB  OR  BACILLUS  OF  BLUE-GREEN  Pus. 

First  described  in  1872  by  Schroeter.     Found  in  green  pus,  and  is  widely  distributed  in  nature. 

REFERENCES.     Schroster:  Cohn's  Baitraege  zur  Biologie,  1872,  1;  126.     Barker:  Jour.  Am.  Med.  Asso.,  1897,  July  31.     Jordan:  Jour. 
Exp.  Med.  1899,    627.     Lartigan,  Ibid.,  1898;  595;  A.  287;  H.  138;  L.  &  K.  120;  M.  &  R.  170;  M.  &  W.  160;  McF.  197;  P.  535;  S.  454. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 

Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

b   Loeffler's  methylen-blue  

5    Motility                

7     Special  characters,  such  as:  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature: . 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.:.. . . 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  72  hours percent., hours. 

(5)    gas  formula,  H  :  COS  :  :  : 


per  cent. 


.  to  ammonia  . 


.days, 
.days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[130] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium, 
Incubation 
Temp.  (°C) 

24           HOURS 

48           HOURS 

6             DAYS 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 

Colonies. 

* 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

1 

/\ 

/      \ 

I          \ 

(4) 

Agar 
Streak. 

- 

^ 

A 

/  \ 

/  \ 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

^ 

(7) 

Special 

Media. 

^ 

[131] 


EXERCISE  XCVI.    MICROSPIRA  COMMA  (Koch)  Schroetei-. 

COMMA  BACILLUS;  CHOLERA  VIBRIO. 

First  described  by  Koch  in  1884.  Found  in  the  intestinal  contents  of  cholera  patients  and  has  also  been  isolated  several  times  from 
a  water  supply. 

REFERENCES.  Koch:  Berl.  Klin.  Wochenschr.  1884,  no.  31  u.  32:  A.  401;  H.  244;  L.  &  K.  181;  M.  &  R  402;  M.  &  W.  152;  McF.  311; 
P.  508;  S  500. 


MORPHOLOGICAL  CHARACTERS. 

en 

B 

VM    = 

0~ 
V  3 

BU 

Incubation 
temp.  (°C.) 

SKETCHES. 

i;  Form: 

c   Gelatin                                                                 

5    Motility                                

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  :. .. 


z.     Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: . 

4.  Figment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

6.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours ,48  hours 

9.  Enzyme  production  :  proteoly tic 


....(2)    closed  arm: 

per  cent.,  72  hours percent., hours. 

..(5)    gas  formula,    H  :  CO2  :  :  : 


per  cent. 


,  to  ammonia. 


.  days. 
days. 


.  diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[132] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium. 
Incubation 
Temp.  (°C) 

24       .      HOCES. 

48  HOURS. 

0  ....     DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

• 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

* 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

/\ 

i\ 

(4) 

Agar 
Streak. 

A 

f  \ 

v            / 

A, 

(5) 
Potato. 

(6) 

\ 

Bouillon. 

^ 

- 

(7) 

Special 

Media. 

[133] 


EXERCISE  XCVII.    MICROSPIRA  flETSCHNlKOVI  nig. 

VIBRIO  METSCHNIKOVI. 

First  described  in  1888  by  Gamaleia.     Found  in  intestinal  contents,  blood  and  organs  of  chickens  suffering  from  a  disease  resem- 
bling chicken  cholera. 

REFERENCES.     Gamaleia:  Ann.  d  1'  Inst,  Past.   1888,  2;  482.     A.  441 ;  H.  256;  M.  &  R.  436;  McF.  332;  P.  593;  S.  511. 


MORPHOLOGICAL  CHARACTERS. 

E 
'oS 

w'a 

& 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 
a.  Bouillon  r  

b.  Agar  ,.  .. 

c   Gelatiu                        

d   Other  media              

2   Size                                   

b.  Loeffler's  methylen-blue  

• 

5.  Motility:  

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light ,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)     open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates:  to  nitrites 

8.  Indol  production;  24  hours ,  48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production:  protcolytic 


...(2)  closed  arm: , 

.percent.,  72  hours percent., hours percent. 

...(5)    gas  formula,   H  :  CO«  :  :  : 


to  ammonia., 


days. 
days. 


.  diastatic. 


10.  Characteristic  odor., 

11.  Pathogenesis 


[134] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium. 
Incubation 
Temp.  (°C) 

24           HOURS 

48      .  .  HOURS 

C  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

• 

(3) 

Gelatin 

Stab. 

A 

i 

A 

A 

(4) 

Agar 
Streak. 

A 

/\ 

^    / 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

Media. 

V   } 

[135] 


EXERCISE  XCVIII.    fUCROSPIRA  FINKLERI  Schroeter. 

SPEILLUM  OF  FINKLER  AND  PRIOR. 

First  described  in  1884  by  Finkler  &  Prior.     Deutsche  Med.  Wochenschr.,  1884,  632. 
REFERENCES.     A.  429;  H.  257;  M.  &  R.  428;  McF.  326;  P.  589;  S  509. 


MORPHOLOGICAL  CHARACTERS. 

B 
oj 

0»"p 

1" 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin  . 

d   Other  media 

2   Size 

5.  Motility  :  

b.  Flagella  stain  

6.  Spores  

7.  Special  characters,  such  as:  ."  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: , 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (:)  open  arm: 

(3)  rate  of  development:  24  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production ;  24  hou  rs 

fecal  odor;  24  hours 

9.  Enzyme  production :  proteoly tic 


per  cent.,  48  hours. 


.  ..(2)  closed  arm: 

per  cent.,  72  hours percent hours per  cent. 

..•(5)    gas  formula,    H  :  COj  :  :  : 


,  to  ammonia. 


,  48  hours. . .. 
,  48  hours 


.  days 
.  days  . 


diastatic 


10.  Characteristic  odor. 

11.  Pathogenesis 


[186] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium, 
Incubation 

24            HOURS 

48             HOURS 

(i  DAYS. 

SKETCHES. 

Temp.  ("C) 

(1) 

Gelatin 

plate  : 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(2) 

Agar 

> 

plate: 

(a)   Surface 

Colonies. 

(b)  Deep 

Colonies. 

(3) 

Gelatin 

Stab. 

L 

u 

(4) 
Agar 

A 

A 

Streak. 

1 

1 

! 

^ 

V                J 

(5) 

A 
/    \ 

,A 

/        \ 
/          i 

1      \ 

Potato. 

J 

(6) 
Bouillon. 

^ 

^ 

(7) 

Special 

Media. 

^ 

^^ 

[137] 


Name  of  organism 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 
Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

2   Size 

a.  Aqueous  gentian-violet  1  

b.  Loeffler's  methylen-blue  

5    Motility                 

• 

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.     Relation  to  temperature: . 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.:.. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:      (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours ,48  hours , 

fecal  odor;  24  hours ,48  hours , 

g.    Enzyme  production:  proteolytic 


(2)     closed  arm: 

.per  cent.,  72  hours per  cent., 

, (5)    gas  formula,  H  :  COS  :  : 


.  hou  rs per  cent . 


,  to  ammonia 


.days. 

.days. 


.diastatic . 


10.  Characteristic  odor 

11.  Pathogenesis '. . 


[188] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium. 

24          HOURS 

48  HOURS. 

6  ...      DAYS 

SKET 

'  '! 

ES 

Incubation 

Temp.  (°C) 

(1) 

Gelatin 

plate  : 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(2) 

Agar 

plate  : 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

• 

(3) 

Gelatin 

Stab. 

L 

U 

(4) 
Agar 

A 

A 

/         \ 

Streak. 

u 

^          / 

(5) 

A 

,/\ 

A 

Potato. 

U 

U 

(6) 
Bouillon. 

Special 

Media. 

- 

[139] 


Name  of  organism . . 
Source,  habitat,  etc. 


References 


MORPHOLOGICAL  CHARACTERS. 


SKETCHES. 


Form: 

a.  Bouillon. 


*.  Agar  . . . . 
c.  Gelatin . . 


d.  Other  media 

2.  Size 

3.  Cell  groupings 

and  arrangements 

in  growths 

4.  Staining  powers: 

a.  Aqueous  gentian-violet.. 
*.  Lot-filer's  methylen-blue. 

c.  Gram's  stain 

d.  Special  stains 

5.  Motility .' 

a.  Character  of  movement  . 

b.  Flagella  stain 

6.  Spores 


7.  Special  characters,  such  as: 

deposits,  vacuoles 

pleomorphic  and  involution  forms,  capsules,  etc. 


PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  :.. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Figment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteoly tic 


....(2)    closed  arm:    

per  cent.,  72  hours per  cent 

..(5)    gas  formula,    H  :  CO2  :  : 


,  hours per  cent. 


,  to  ammonia . 


.  days, 
days. 


.  diastatic. 


10.    Characteristic  odor 

it.    Pathogenesis '. ... 


[140] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  (°C) 

24           HOURS 

48  ....    HOURS 

C      .      DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3)- 

• 

Gelatin 

Stab. 

J 

A 

/\ 

(4) 

Agar 
Streak. 

A 

,'  \ 

>>         y 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

• 

Media. 

[141] 


Name  of  organism  . . 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

M 

_£ 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                 

- 

• 

PHYSIOLOGICAL  CHARACTERS. 

i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates:  to  nitrites 

8.  Indol  production:  24  hours ,48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


...(2)  closed  arm: 

.percent.,  72  hours per  cent., 

...(5)    gas  formula,   H  :  COa  :  : 


.hours percent. 


to  ammonia. 


days. 
days. 


.  diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[142] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium 
Incubation 
Temp.  (°C 

24           HOURS 

48  HOURS. 

G  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

(4) 
Agar 

Streak. 

* 

A 

1  \ 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

Media. 

[143] 


Same  of  organism  . . 
Source,  habitat,  etc. 


References 


MORPHOLOGICAL  CHARACTERS. 

m 
D 

k. 

4)*a 

1" 

Incubation 
temp.  ("C.) 

SKETCHES. 

i.  Form: 

d.  Special  stains  

5.  Motility:  .             

b.  Flagella  stain  

- 

6.  Spores  

7.  Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 

fecal  odor;  24  hours 

9.  Enzyme  production :  proteolytic 


(2)  closed  arm: 

per  cent.,  48  hours per  cent.,  72  hours per  cent.,  hours per  cent. 

(5)    gasformula,    H  :  CO,  :  :  : 


,  to  ammonia. 


.,  48  hours. 
. . ,  48  hours. . 


.  days 
.  days  . 


diastatic 


10.  Characteristic  odor. 

11.  Pathogenesis 


[144] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  (°C) 

24            HOURS 

48  HOURS 

(i  .  .        DAYS 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

• 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

(4) 
Agar 

Streak. 

*v  X 

A 

A 

^         / 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

- 

(7) 

Special 

Media. 

^ 

^ 

[145] 


Name  of  organism 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

Age  of 

Cultures. 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin  .                  

d   Other  media                  

4.  Staining  powers:  

• 

a.  Aqueous  gentian-violet  

b,  Loeffler's  methylen-blue  -  

c.  Gram's  stain  

d.  Special  stains  

5.  Motility:  

a.  Character  of  movement  

* 
b.  Flagella  stain  

6.  Spores  

• 

PHYSIOLOGICAL  CHARACTERS. 


i.     Relation  to  temperature: 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.     Gas  production  in  glucose  media: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites  

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours ,48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  72  hours per  cent 

(5)    gas  formula,  H  :  COS  :  : 


.hours percent. 


,  to  ammonia  . 


.days. 
. days . 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[14C] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium. 
Incubation 
Temp.  (°C) 


24  .     , .  HOURS. 


48. .       .  HOURS. 


DAYS. 


SKETCHES. 


(1) 

Gelatin 
plate : 

(a)  Surface 
Colonies 

(b)  Deep 
Colonies. 


(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 


(3) 

Gelatin 
Stab. 


(4) 

Agar 
Streak. 


(5) 


Potato. 


(6) 


Bouillon. 


(?) 

Special 

Media. 


A 


A 


A 


[147] 


CHAPTER  VII. 

PATHOGENIC  ANAEROBES. 


Anaerobic  bacteria  may  be  furnished  conditions,  which  permit  of  their  development, 
in  a  variety  of  ways  and  a  very  considerable  number  of  pieces  of  apparatus  have  been 
devised  to  secure  this  end.  In  a  general  way  all  of  the  methods  may  be  grouped  under 
the  following  heads: 

1.  Displacement  of  air. 

2.  Absorption  of  oxygen. 

3.  Exhaustion  of  air. 

4.  Exclusion  of  air. 

5.  Miscellaneous  methods,  in  the  presence  of  reducing  substances  as  litmus,  or 
a  strongly  aerobic  germ,  etc. 

The  first  two  methods  are  the  most  reliable.  In  the  displacement  method,  hydro- 
gen, carbon  dioxide  or  illuminating  gas  may  be  used;  hydrogen  is  best.  This  gas  is 
readily  prepared  by  the  action  of  sulphuric  acid  (1:8)  on  zinc.  Either  a  Kipp  generator 
may  be  used  or  one  of  a  simpler  construction.  The  gas  should  be  washed,  1st.  in  lead 
nitrate  to  absorb  the  sulphuretted  hydrogen,  2nd.  in  silver  sulphate  to  absorb  any 
arseniuretted  or  phosphuretted  hydrogen,  and  3rd.  in  potassium  hydrate  to  remove  sul- 
phur and  carbon  dioxide. 

The  cultures  are  made  in  media  containing  glucose  (which  should  preferably  be 
freshly  prepared  and  always  boiled  immediately  before  being  inoculated),  either  as  test- 
tube  or  plate  cultures.  Novy's  anaerobic  jars  are  perhaps  the  most  satisfactory  recep- 
tacles for  the  cultures.  (For  careful  description  of  same,  see  N.  306.) 

In  the  second  method  (Buchner's  method)  an  alkaline  solution  of  pyrogallic  acid 
is  used  to  absorb  the  oxygen.  The  cultures  may  be  placed  in  Novy  jars  or  similar  re- 
ceptacles ;  for  tube  cultures  a  large  wide  mouthed  bottle  fitted  with  a  rubber  cork  does  very 
well.  The  dry  pyrogallic  acid  is  placed  in  the  bottom  of  the  receptacles,  about  1  gram 
to  every  100  cc-  of  air  space,  the  tubes  are  put  in  place,  then  about  10  cc.  of  a 
normal  sodium  hydroxide  is  added  to  each  gram  of  pyrogallic  acid,  and  the  apparatus 
immediately  and  hemetically  sealed. 

REFERENCES.  A.  206;  L-  &  K.  98;  M.  &  R.  68;  M.  &  W.  117;  McF.  153;  P. 
233;  S.  78. 


EXERCISE  XCIX.     BACTERIUM  WELCHII  Mig. 

BACILLUS  AEROGENES  CAPSULATUS. 

First  described  by  Welch  in  1892.  Occurs  at  autopsies  in  which  gas  bubbles  are  present  in  the  larger  vessels,  accompanied  by  the 
formation  of  numerous  small  cavities  in  the  liver  containing  gas.  It  has  been  found  also  in  emphysematous  phlegmons,  in  puerperal 
sepsis,  in  peritonitis  and  in  other  conditions  (M.  &  W.).  Widely  distributed  in  nature.  (Welch.) 

REFERENCES.  Welch  and  Nuttall:  Bull.  Johns  Hopkins  Hospital,  1892,  8;  81;  Welch  &  Flexner:  Jour.  Exp.  Med.,  1896,  1;  5;  H.  140; 
M.  &  W.  173;  McF.  463;  P.  545;  S.  731. 


MORPHOLOGICAL  CHARACTERS. 

i 

"32 

IB 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin 

5    Motility                                                          .                                                                                            .           ....            

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:.. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

'desiccation,  light,  disinfectants,  etc.:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours percent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates:  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hou  rs ,  48  hours 

9.  Enzyme  production  :  proteolytic 


...(2)  closed  arm: 

.per  cent.,  72  hours per  cent., 

...(5)    gas  formula,  H  :  CO«  :  : 


—  hours.. 


percent. 


to  ammonia.. 


days, 
days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[150] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 
Incubation 
Temp.  (»C) 

24            HOURS 

48  HOUES. 

G  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

A 

A 

(4) 

Agar 
Streak. 

A 

f  \ 

V                / 

A 

(5) 
Potato. 

• 

(6) 

Bouillon. 

' 

- 

- 

(7) 

Special 

Media. 

[151] 


EXERCISE  C.     BACILLUS  CHAUVAEI  Arloing,  Cornevin  and  Thomas. 

BACILLUS  OF  SYMPTOMATIC  ANTHRAX. 

First  described  by  Arloing,  Cornevin  and  Thomas  in  1887.     It  occurs  in  the  subcutaneous  tissue,  muscles  and  serous  exudate  of  ani- 
mals suffering  from  sj'mptomatic  anthrax. 

REFERENCES.     Arloing,  Cornevin  and  Thomas ;  Le  Charbon  symptomatique  du  baeuf ,  2nd  edit.  Paris,  1887 ;  A.  482 ;  H.  304 ;  McF. 
453;  P.  503 ;  S.  403. 


MORPHOLOGICAL  CHARACTERS. 

i 

•32 
f 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

b.  Agar  

d   Other  media  . 

2   Size. 

4.  Staining  powers:  .     .  .             T  

5.  Motility: 

a.  Character  of  movement  

b.  Flagella  stain  

6.  Spores  

7.  Special  characters,  such  as:  

deposits,  vacuoles  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature:. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as : 

desiccation,  light,  disinfectants,  etc: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  medte: 

a.  Shake  culture 

b.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 

fecal  odor;  24  hours 

9.  Enzyme  production :  proteolytic 


per  cent.,  48  hours. 


.  ..(2)  closed  arm: 

per  cent.,  72  hours per  cent., 

•  ••(5)    gas  formula,    H  :  COj  :  : 


hours. 


per  cent. 


,  to  ammonia . 


,  48  hours. 
,  48  hours.. 


.  days 
days  . 


.  diastatic  . 


10.  Characteristic  odor. 

11.  Pathogenesis 


[152] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium, 

24             HOURS 

48    ...  HOURS. 

fl  .  .  .      DAYS 

SKEI 

ITU 

JES 

Incubation 
Temp.("C) 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

L/ 

u 

(4) 

Agar 
Streak. 

A 

i 

/\ 
/   \ 

/       \ 

- 

^ 

^         y 

(5) 
Potato. 

A 
/    \ 

/          t 

A 

(6) 

Bouillon. 

^ 

^_^ 

(7) 

Special 

Media. 

^ 

^^ 

[153] 


EXERCISE  Cl.     BACILLUS  OEDEMATIS  Liborius. 

BACILLUS  OF  MALIGNANT  OEDEMA. 

First  described  by  Pasteur  in  1877.     Widely  distributed  in  soil  and  putrefying  material.     Few  cases  on  record  of  infection  of  man. 
REFERENCES.     Z.  f.  H.,  1886;  1 : 158;  A.  476;  H.  302;  L.  &  K.  305;  M.  &  R.  :!94;  M.  &  W.  175;  M.  &  W.  459;  P.  543;  S. 


MORPHOLOGICAL  CHARACTERS. 

• 
£ 
"32 

$ 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

c   Gelatin                                    .       .                     ...                                                                                     

a.  Aqueous  gentian-violet  

b,  Loeffler's  methylen-blue  

5.  Motility:     

a.  Character  of  movement  

pleomorphic  and  involution  forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature: . 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 
4-    Pigment  production: 


i.    Gas  production  in  glucose  media: 

a.  Shake  culture 

A.  Fermentation  tube,  growth  in:     (i)    open  arm: 

(3)  rate  of  development:    24  hours per  cent.,  48  hours. 

(4 )  reaction  in  open  arm : 

6.  Acid  or  alkali  production,  litmus  milk  

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production:  proteolytic 


(2)    closed  arm: 

.per  cent.,  72  hours per  cent., 

(5)    gas  formula,  H  :  COS  :  : 


. .  ..hours. 


per  cent. 


. ,  to  ammonia 


.days, 
.days. 


.diastatic. 


10.  Characteristic  odor. 

11.  Pathogenesis 


[154] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium, 
Incubation 

24           HOURS 

48  HOURS 

6  .  .        DAYS 

SKETCHES. 

Temp.  (°C) 

(1) 

Gelatin 

plate  : 

(a)  Surface 

•  t 

Colonies. 

(b)  Deep 

Colonies. 

(2) 

Agar 

plate: 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(3) 

Gelatin 

, 

Stab. 

o 

0 

(4) 
Agar 

A 

A 

Streak. 

i 

• 

*                 . 

V^  ^^ 

(5) 

A 

A 

Potato. 

b; 

^ 

(6) 
Bouillon. 

' 

o 

^ 

(7) 

Special 

Media. 

^  ' 

v  y 

[155] 


EXERCISE  Cll.     BACILLUS  TETANI  Nicolaier. 

Discovered  by  Nicolaier,  1884.  First  cultivated  by  Kitasato,  1889.     Occurs  in  man  and  animals  suffering  from  the  disease  and  widely 
distributed  in  nature,  especially  in  soil. 

REFERENCES.     Nicolaier :  Deutszche  Med.     Wochenschrift,  1884;      Kitasato:   Deutsche  Med.   Wochenschrift,  1889;      A.  469;   H.  290; 
L.  &  K.  230;  M.  &  R.  376;M.&W.  171;  McF.  274;P.  385;  S.  482. 


MORPHOLOGICAL  CHARACTERS. 

tfi 

<y 

u 

p 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

2    Size          -                

5    Motility 

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  :. 


2.     Relation  to  tree  oxygen: 

j.    Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 
4.     Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  i:i:    ui    open  .inn: 

(3)  rate  of  development:  24  hours per  cent.,  48  luurs 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus    milk 

j.    Reduction  of  nitrates;  to  nitrites 

8.    Indol  production;  24  hours ,  48  hours 

fecal  odor;  24  hours 48  hours 

g.    Enzyme  production  :  proteoiytic 


....(2)    closed  arm:   

.  per  cent.,  72  hours per  cetit., 

...(5)    gas  formula,    H  :  CO^  :  : 


....  hours. 


per  cent. 


,  to  ammonia . 


.  days. 
.  days. 


.  diastatic . . . 


10.  Characteristic  odor. 

11.  Pathogenesis 


[156] 


CULTURE  CHARACTERS. 


Reaction 
of  Medium 

24  .  .     HOURS. 

48  ...      HOURS 

6             DAYS 

SKET 

MI 

ES 

Incubation 
Temp.  (°C; 

(1) 

Gelatin 

plate: 

(a)  Surface 
Colonies 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

U 

U 

(4) 

Agar 
Streak. 

A 

A 

A 

P) 

Potato. 

• 

A 

A 

A 

(6) 

Bouillon. 

^^ 

^^ 

(7) 

Special 

Media. 

• 

^    ) 

[157] 


Name  of  organism  . , 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

*°s 

Incubation 
temp.  (oC.) 

SKETCHES. 

i.  Form: 
a.  Bouillon  *  

b    Agar.            

c  Gelatin           

d  Other  media        

2   Size                                  

in  growths  

c.  Gram's  stain  

5    Motility 

b    Flagella  stain 

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature  : 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc.: 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (i)    open  arm: 

(3)  rate  of  development:  24  hours per  cent.,  48  hours. 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production:  24  hours ,48  hours 

fecal  odor;  24  hours 48  hours 

9.  Enzyme  production  :  proteolytic 


...(2)  closed  arm: 

.  per  cent.,  72  hours per  cent 

...(5)    gasformula,  H  :  CO«  :  : 


.  hours per  cent. 


to  ammonia.. 


days, 
days. 


.diastatic. 


10.    Characteristic  odor, 
n.    Pathogenesis 


[158] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium. 
Incubation 

24           HOURS 

48  HOURS. 

0  DAYS. 

SKETCHES. 

(1) 

Gelatin 

plate  : 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(2) 

Agar 
plate: 

(a)  Surface 
Colonies. 

(b)  Deep 
Colonies. 

(3) 

Gelatin 

Stab. 

A 

i 

A 

A 

(4) 

Agar 
Streak. 

A 

,'  ^ 

i.         y 

A 

(5) 
Potato. 

(6) 

Bouillon. 

- 

- 

(?) 

Special 

Media. 

^ 

^^ 

[159] 


Name  of  organism  . . 
Source,  habitat,  etc. 


MORPHOLOGICAL  CHARACTERS. 

i 

"32 

J3 

Incubation 
temp.  (°C.) 

SKETCHES. 

i.  Form: 

< 

c  Gelatin 

2   Size.                     

b.  Loeffler's  methylen-blue. 

5    Motility  • 

a.  Character  of  movement  . 

7.  Special  characters,  such  as:  ..  . 

pleomorphic  and  involution 

forms,  capsules,  etc  

PHYSIOLOGICAL  CHARACTERS. 


i.    Relation  to  temperature-.. 


2.  Relation  to  free  oxygen: 

3.  Relation  to  other  agents,  such  as: 

desiccation,  light,  disinfectants,  etc:. 

4.  Pigment  production: 


5.  Gas  production  in  glucose  media: 

a.  Shake  culture 

*.  Fermentation  tube,  growth  in:    (i)  open  arm: 

(3)  rate  of  development:  24  hours 

(4)  reaction  in  open  arm: 

6.  Acid  or  alkali  production,  litmus  milk 

7.  Reduction  of  nitrates;  to  nitrites 

8.  Indol  production;  24  hours 

fecal  odor;  24  hours 

9.  Enzyme  production :  proteolytic 


per  cent.,  48  hours. 


—  (2)  closed  arm: 

per  cent.,  72  hours per  cent hours. 

...(5)    gas  formula,    H  :  CO8  :  :  : 


per  cent. 


,  to  ammonia. 


,  48  hours. 
,  48  hours.. 


.  days 
.  days  . 


.  diastatic  . . . 


10.  Characteristic  odor 

11.  Pathogenesis 


[160] 


CULTURE  CHARACTERS. 


Reaction 

of  Medium 
Incubation 

24      .     HOURS 

48  HOURS 

6            DAYS 

SKETCHES. 

Temp.  (°C) 

(1) 

Gelatin 

plate: 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(2) 

Agar 

plate  : 

(a)  Surface 

Colonies. 

(b)  Deep 

Colonies. 

(3) 

Gelatin 

Stab. 

• 

^ 

^ 

(4) 
Agar 

A 

/\ 

Streak. 

i 

u 

v        y 

(5) 

A 
/    \ 

A 

/        \ 

/  \ 

Potato. 

.          i 

^.,^s 

^ 

(6) 
Bouillon. 

Special 

Media. 

[161] 


CHAPTER  VIII. 

ANIMAL  INOCULATION  AND  STAINING  OF  BACTERIA  IN  TISSUE. 


EXERCISE  CIII.     ANiriAL  INOCULATION. 

METHODS  OF  INOCULATION.  Animal  inoculation  is  practiced  to  determine  the  path- 
ogenic properties  of  an  organism  and  also  the  character  of  the  tissue  changes  produced. 
The  animals  commonly  iised  are  white  mice  and  rats,  rabbits,  guinea  pigs  and  pigeons. 
Inoculations  are  usually  made  intraperitoneally,  intravenously  or  subcutaneously,  and 
in  special  cases  into  the  pleural  cavity,  brain,  eye,  etc.,  etc.  Mice  require  a  holder,  the 
inoculation  being  made  at  the  root  of  the  tail.  Other  animals  can  usually  be  held  by  an 
assistant. 

Subcutaneous.  The  place  selected  is  usually  the  abdominal  wall.  Pigeons  are 
inoculated  in  the  pectoral  muscles;  the  hair  or  feathers  should  be  removed  and  the  skin 
washed  with  a  disinfectant,  e.  g. ,  5%  carbolic  acid. 

a.  For  liquids  a  sterilized  hypodermic  syringe  is  used.     A  fold  of  the  skin  is  raised, 
the  needle  of  the  syringe  inserted  and  the  requisite  amount  of  culture  injected. 

b.  For  solid  material  a  pocket  is  made  which  is  stitched,  or  sealed  with  contractile 
collodion,  after  the  material  is  introduced. 

Intraperitoneal.  Prepare  seat  of  inoculation  as  above,  then  plunge  needle  directly 
into  the  peritoneal  cavity. 

Intravenous.  A  rabbit  is  generally  chosen  for  this  purpose  and  the  inoculation  made 
into  the  dorsal  vein  of  the  ear.  Slight  pressure  at  the  base  of  the  ear  will  render  the 
vein  more  prominent.  Avoid  the  introduction  of  air,  which  causes  immediate  death,  and 
keep  the  animals  under  close  observation  for  one  hour. 

Inoculation  into  Lymphatic  system.  Fluid  cultures  or  suspensions  of  bacteria  can 
be  injected  into  the  lymphatics  by  way  of  the  testicles,  by  plunging  the  point  of  the 
needle  into  the  substance  of  the  testicle  and  injecting  the  desired  amount  of  fluid- 

Inoculation  into  the  Pleural  Cavity.  Where  necessary  the  needle  is  introduced  into 
the  pleural  cavity  between  the  ribs.  It  is  very  difficult  to  perform  this  experiment  with- 
out injuring  the  lung. 

Inoculation  into  the  Anterior  Chamber  of  the  eye.  Rarely  practiced.  The  eye  is 
treated  with  a  few  drops  of  cocaine  (2  %  solution)  and  then  the  needle  is  inserted 
through  the  cornea  just  in  front  of  its  junction  with  the  sclerotic,  the  needle  passing 
into  the  anterior  chamber  in  a  plane  parallel  to  the  plane  of  the  iris. 

The  following  inoculations  are  those  most  frequently  made: 

Streptococcus  pyogenes.     Mice  or  rabbits,  intravenous. 

Sarcina  tetragena.     Guinea  pigs  and  white  mice,  subcutaneous- 
Bacterium  anthracis.     Guinea  pigs  or  rabbits,  subcutaneous. 

pneumoniae.     Rabbits  and  mice,  subcutaneous. 

-  pnewmonicum.     Mice  and  young  rats,  intraperitoneal. 

tuberculosis.     Guinea  pigs,  rabbits  and  field  mice,  any  method  of  inoculation 

will  produce  the  disease. 


164  Medical  Bacteriology. 

B.  mallei.     Male  guinea  pigs,  infection  of  lymphatics. 

—  (liphtheriae.     Guinea  pigs,  rabbits  and  fowl,  subcutaneous  and  intratracheal . 
Bacillus  pestis.     Bats,  mice,  guinea  pigs  and  rabbits,  subcutaneous. 

—  Kiripestifer.     Rabbits  and  mice,  subcutaneous. 

STERILIZATION  OF  INSTRUMENTS.  These  are  best  sterilized  by  boiling  in  a  solution 
of  soda  or  borax  for  15  minutes.  This  is  accomplished  in  an  especially  designed  ap- 
paratus  or  in  an  ordinary  enamel  stew  pan.  In  case  of  emergencies  the  instruments 
may  be  dipped  in  benzene  or  alcohol  and  burned.  This  is  less  injurious  to  the  instru- 
ment than  heating  in  the  direct  flame. 

Use  blank,  p.  168,  for  preservation  of  data. 

OBSERVATION  OF  INOCULATED  ANIMALS.  After  inoculation  the  animals  should  be 
placed  in  separate  cages,  or  if  placed  together  they  must  be  described  or  marked  so  as 
to  be  easily  identified.  They  must  also  be  kept  under  constant  observation  and  the 
following  conditions  noted: 

1.  Temperature. 

2-  Loss  of  Weight. 

3.  Peculiar  position  in  cage. 

4.  Loss  of  appetite. 

5.  Condition  of  the  coat  or  hair. 

6.  Condition  of  the  secretion  of  the  air  passages,  conjunctiva  and  kidneys;  diarrhea 
or  hemorrhage  from  the  bowels. 

7.  The  condition  of  the  seat  of  inoculation. 

The  animals  should  be  fed  regularly,  weighed  at  the  same  hour  each  day  and  the 
temperature  taken  at  the  rectum. 

POST  MORTEM  EXAMINATION. 

Perform  the  autopsy  as  soon  as  possible  after  death.  When  delay  cannot  be 
avoided,  place  the  animal  in  the  ice- chest  until  such  time  as  is  convenient. 

A. 

1.  Inspect  externally  and  note  presence  and  character  of  any  lesion. 
2-   Sterilize  a  suitable  post-mortem   board  with  corrosive  sublimate  solution,   1   to 
1000,  place  the  animal  belly  upwards  and  tack  the  four  legs  fast  to  the  board. 

3.  Wash  the  surface  of  the  thorax  and  abdomen  with  corrosive  sublimate  solu- 
tion, make  an  incision  through  the  skin  at  the  pubis,  introducing  one  blade  of  the    scis- 
sors, and  extend  the  incision  as  far  as  the  chin. 

4.  Carefully  dissect  the  skin  away   from  the   abdomen,  thorax,  axillary,  inguinal, 
and  cervical  regions,  and  fore  and  hind  legs,  and  pin  it  to  the  board  as  far  as  possible 
from  the  thorax  and  abdomen.     It  is  from  the  skin  that  the  chances  of  contamination 
are  greatest. 

B. 

All  incisions  from  now  on  are  made  with  sterilized  instruments. 

1.  Take  an  ordinary  potato-knife,  heat  it  quite  hot,  and  place  it  on  the  abdomen  in 
the  region  of  the  linea  alba  until  the  fascia  begins  to  burn ;  the  knife  is  then  held  trans- 
versely to  this  line  over  the  center  of 'the  abdomen,  making  two  sterilized  tracks  through 
which  the  abdomen  may  be  opened  by  crucial  incisions:  two  burned  lines  are  also  made 
along  the  sides  of  the  thorax. 


166  Medical  Bacteriology. 

2.  Make  a  central  longitudinal  incision  from  the  sternum   to  the  genitalia   with 
sterile  scissors,  the  abdominal  wall  being  held  up  with  sterilized  forceps,   or  a  hook  to 
prevent  the    viscera  being   injured.     A  transverse  incision  is  made  in  a  similar  man- 
ner.    Cut  through  the  ribs  with  strong  sterilized  scissors   along  the  sterilized  tracks  on 
the  sides  of  the  thorax,  when  the  whole  anterior  wall  of  the  thorax  is  easily  lifted  and 
entirely  removed  by  severing  the  diaphragm  connections. 

3.  When  the  thoracic  and  abdominal  cavities  are  fully  exposed,  a  careful  examina- 
tion of  the  organs  and  surroundings  is  made  without  disturbing  them. 

Culture  plates  (Petri-dish)  or  roll  cultures  are  prepared  from  the  blood,  liver,  spleen, 
kidneys,  and  any  exudates  present. 
The  method  is  as  follows: 

(1)  Heat  a  scalpel  and  scorch  a  small  surface  of  the  organ  from  which  the  cultures 
are  to  be  made. 

(2)  Heat  the  scalpel  again  and  penetrate  the  capsule  of  the  organ  with  the  point, 
and  through  the  opening  insert  a  stout  sterilized  platinum  loop,  push  it  into  the  tissues, 
twist  around,  and  obtain  enough  material  from  the  center  of  the  organ  to  make  the 
culture. 

Cultures  from  blood  are  usually  made  from  one  of  the  heart  cavities,  the  surface 
being  seared  with  a  hot  knife  before  opening.  As  soon  as  the  culture  material  is  ob- 
tained, cover-glass  specimens  are  prepared  from  each  organ  and  existing  exudates. 

Small  pieces  of  each  organ  are  also  preserved  for  future  examination. 

When  the  autopsy  is  finished  the  remainder  of  the  animal  should  be  burned  and  the 
instruments  should  be  sterilized.  Wash  the  post-mortem  board  with  sublimate  solution. 
The  cover- glasses  and  other  material  likely  to  contain  infectious  matter  must  also  be 
sterilized  when  of  no  further  use. 

Cultures  are  to  be  incubated  at  38°  C.,  growth  examined  microscopically,  and  by 
means  of  sub-cultures. 

REFERENCES.  The  above  is  taken  largely  from  Bowhill,  74;  see  also  A.  219;  N. 
260;  and  other  texts. 


168  Medical  Bacteriology. 

BLANK  FOR  ANIMAL  EXPERIMENTS. 

Animal  No 

Experimenter     

Animal Sex Age Weight 

Specimens  received  o'clock M . 

Organs  

Museum  No Slide  No. 

Experiment : 


Died  or  killed  o'clock M. 

Autopsy o'clock M. 

Findings : 


Bacteriological  Examination : 


Histological  Examination: 


170 


Medical  Bacteriology. 


EXERCISE  CIV.     PREPARATION  OF  TISSUE  FOR  EXAMINATION. 

Portions  of  the  diseased  tissue,  removed  at  autopsy,  should  be  cut  into  cubes  hav- 
ing edges  about  5  mm.  long  and  treated  as  follows: 

1) .  FIXING.  Use  15  or  20  times  their  volume  of  95%  alcohol  for  24  hrs.  The  speci- 
mens should  be  placed  on  cotton  to  keep  them  near  the  top  and  the  alcohol  changed 
after  3  or  4  hours,  if  they  are  not  to  be  sectioned  immediately  carry  to  80%  alcohol.  . 

Where  larger  sections  are  desired  they  should  be  left  a  longer  time  in  the  alcohol . 

2) .  PREPARATION  FOR  SECTIONING. 


A. 
Paraffin  Method. 

I 

a.  Absolute    Alcohol 
hours.  | 

b.  Xylene  6-24  hours. 


6-24 


c.  Paraffin  melting  at  50°C. 
and  kept  in  an  oven  or  water- 
bath  at  a  temperature  a  few  de- 
grees above   the  melting   point 
of  the  paraffin. 

I 

d.  Embed.       Pour     melted 
paraffin  into  a  paper  box  or  other 
suitable    receptacle     and      with 
warm    forceps,     arrange    block 
of  tissue  in  proper  position  and 
cool  rapidly  by  plunging  into  cold 
water. 


B. 

Gelloidin  Method. 
I 

a.  Mixture     of     ether 
and  absolute  alcohol  (equal 
parts)  24  hours. 

I 

b.  Thin  celloidin  (about 
6%)    24  hours   to    several 
weeks. 

I 

c.  Thick    celloidin 
(about  12%)  24  hours  to 
several  weeks. 

I 

d.  Remove   block     of 
tissue  to  a  piece   of   wood 
fiber  covered  with  "thick" 
celloidin,  orient,  dry  a  few 
minutes  in  air  then  place  in 
80%  alcohol  for  6-24  hours. 


c. 
Freezing  Method. 

I 

a.  Place    in    1% 
Formalin  2  hours. 

I 

b.  Place  tissue  on 
plate    of    freezing 
microtome  in  water  or 
better  first  soak  tissue 
in  a  syrupy  solution 
of    gum-arabic    and 
moisten   plate   with 
same  before  freezing. 


I 

3).  SECTIONING.     Cut  sections  from  10-12  /*  thick. 
4).  MANIPULATION  OP  SECTIONS. 

a.  Celloidin  sections   can  be  preserved  in  80  %  alcohol  and  are   best   stained  by 
placing  the  sections  first  in  water  and  then  in  the  stain-     The  various  reagents  are  best 
used  in  watch  glasses  and  the  sections  transferred  from  one  to  the  other  by  means  of  a 
section  lifter. 

b.  Paraffin  sections  should  be  fixed  to  the  slide  or  cover- glass  as  follows:     A  water- 
bath  is  heated  up  to  a  few  degrees  below  the  melting  point  of  the  paraffin,  the  sections 
are  placed  on  the  water  'where  they  will  straighten  out  and  are  then  transferred  to  the 
slide  or  more  conveniently  to  the  cover-glass  by  simply  dipping  the  same  into  the  water 
and  drawing  up  the  section  by  means  of  the  fine  point  of  a  pair  of  forceps  or  a  needle, 
draining  off  the  water  and  drying  the  section  in  an  incubator  for  a  few  hours-     The  sec- 
tions are  more  secure  if  the  cover-glasses  are  first  smeared  with  a  thin  coat  of  egg 
albumin.     When  the  sections  are  once  fixed  to  the  cover  the  staining  can  be  carried  on 
in  the  forceps  as  with  ordinary  cover- glass  preparations.     Before  staining,  however,  the 
paraffin  must  be  removed;  this  is  done  with  xylene  and  this  in  turn  removed  with  absolute 
alcohol. 

REFERENCES-     A.  173;  M.  &  W.  204-239;  N.  531. 


172  Medical  Bacteriology. 

EXERCISE  CV.    STAINING  SECTIONS. 

GENERAL  HISTOLOGICAL  METHOD. 
Hcematoxylin  and  Eosin. 

a.  Transfer  sections  from  alcohol  to  distilled  water. 

b.  Stain  in  aluin-ha8inatox\'lin  2,  5  to  30  minutes.     The  stain  may  be  prepared  as 
follows  (Boehmer): 

1.  Haematoxylon  crystals,  -                                                                       1  gram. 
Absolute  alcohol,  -  -  10  cc. 

2.  Alum,  -  20  grams. 
Distilled  water,  -  200  cc. 

Cover  the  solutions  and  allow  them  to  stand  over  night.  The  next  day  mix  them 
and  allow  the  mixture  to  stand  for  one  week  in  a  wide-mouthed  bottle  lightly  plugged 
with  cotton.  Then  filter  into  a  bottle  provided  with  a  good  cork.  The  solution  is  now 
ready  for  use  but  its  staining  powers  improve  with  age. 

c.  Wash  the  sections  in  several  changes  of  water  until  they  have  lost  all  traces  of  a 
red  tint. 

d.  Counter- stain  with  eosiu  (iV  to  j  %  in  60  %  alcohol)  1  to  5  minutes. 

e.  Alcohol,  95  %,  two  or  three  changes  to  dehydrate  and  remove  excess  of  counter- 
stain. 

/.  Clear  in  oil  of  origanum  or  Dunham's  mixture,  white  oil  of  thyme  4  parts,  oil 
of  cloves  1  part. 

GENERAL  BACTERIOIX)GICAL  METHODS. 

A.  Loeffler's  Universal  Method. 

a.  Take  sections  out  of  alcohol  into  Loeffler's  methvlen  blue  for  5-30  minutes. 

b.  Decolorize  iu  acetic  acid  (0.  1%)  10  to  20  seconds. 

c.  Dehydrate  in  absolute  alcohol,  two  or  three  changes,  a  few  seconds. 

d.  Clear  in  xylene. 
'e.  Mount  in  balsam- 

B.  Weigert's  Method. 

a.  From  alcohol  to  Ehrlich's  anilin  water  gentian  violet  5-15  minutes. 

b.  Wash  inO.  6%  salt  solution. 

c.  Dry  with  filter  paper. 

d.  Place  in  potassium  iodide  and  iodine  solution   (iodine  1  part,   potassium 
iodide  2  parts,  water  100  parts). 

e.  Dry  with  filter  paper. 

/.  Decolorize  in  a  mixture  of  anilin  oil  2  parts  and  xylene  1  part,  2-5  minutes. 

g.  Clear  in  xylene. 

h.  Mount  in  balsam. 

This  stain  can  only  be  used  with  those  organisms  which  take  the  Gram  stain,  namely: 
8.  pyogenes,  M.  pyogenes,  M.  aureiis,  Sar.  tetmgena,  B.  anthracis,  B.  pneumoniae,  B. 
rhusiopathiae,  B.  tuberculosis,  B:  leprae,  B.  diphtheriae,  P.  aeruginosa,  B.  Welchii,  B. 
chauvaei.  B.  oedematis,  B.  tetani.  and  Strevtothrix  actinomyces. 


174  Medical  Bacteriology. 

SPECIAL  BACTERIOLOGICAL  METHODS. 

Particular  organisms  may  be  stained  as  follows : 
Pyogenic  micrococci.     Loeffler's  or  Weigert's  method. 
Micrococcus  gonorrhoea*,.     Loeffler's  method  gives  the  best  results. 
Sarcina  tetragena,     Loeffler's  or  Weigert's  method. 
Bacterium  unthracis.     Loeffler's  or  Weigert's  method. 
Bacterium  pneumoniae.     Weigert's  method. 

Bacterium  pneumonic-urn.  The  following  method  is  recommended  for  staining  the  cap- 
sules in  sections  (M.  &  W.) : 

a.  Stain  for  24  hours  in  the  incubator  in  the  following  solution: 

Saturated  alcoholic  solution  of  gentian  violet   -  50cc. 

Distilled  water  -       lOOcc. 

Glacial  acetic  acid  lOcc. 

b.  Wash  out  in  1%  solution  of  acetic  acid. 

c.  Alcohol. 

d.  Xylene. 

e.  Canada  balsam. 

Bacterium  cuniculicida.     Loeffler's  Method. 
Bacterium  tuberculosis. 

a.  Weigert's  method    (staining  with  auilin   oil  gentian  violet  24  hours  at  room 
temperature,  or  2-3  hours  at  40°  C.). 

b.  Ziehl-Neelsen's  Method. 

1.  Stain  with  carbol-fuchsin    (12-24  hrs.  room  temperature,  1-3  hrs.  40°  C.) 

2.  Decolorize  with  nitric  acid  (10%)  a  few  seconds  and  then  with  alcohol  (60-90%) 
until  color  is  nearly  all  extracted. 

3-  Counter-stain  with  methylen  blue. 

4.  Dehydrate  with  absolute  alcohol  (a  few  seconds). 

5.  Clear  with  clove  oil. 

6.  Xylene  (and  examine). 

7.  Mount  in  balsam. 

Bacterium  leprae.  This  organism  is  stained  with  the  tubercle  stain,  unless  the  sec- 
tions have  been  kept  in  alcohol  for  some  time,  in  which  case  Weigert's  method  can  be 
employed.  To  differentiate  this  organism  from  B.  tuberculosis,  stain  as  follows: 

a.  An  aqueous  solution  of  fuchsin  6-7  minutes. 

b.  Acid  alcohol  (nitric  acid  1,  alcohol  10)  \  minute. 

c.  Wash  in  water. 

d.  Counter-stain  in  a  saturated  aqueous  solution  of  methylen  blue. 

e.  Alcohol. 
/.    Xylene. 
g.  Balsam. 

The  bacteria  of  leprosy  stain  readily  by  this  method,  tubercle  bacteria  do  not. 
Bacterium  mallei. 
Slow  Method. 

a.  Stain  in  Loeffler's  methylen  blue  6-8  hours. 

b.  Wash  in  distilled  water. 

c.  Tannic  acid  solution  (10  %)  4-5  hours. 


176  Medical  Bacteriology. 

d-  Wash  thoroughly  in  water. 
e.  Dehydrate  in  absolute  alcohol. 
/.  Clear  in  xylene  and  mount. 

Quick  Method. 
«.  Stain  in  carbol-methylen  blue  10-30  seconds. 

b.  Wash  in  distilled  water. 

c.  Tannic  acid  solution  (10  %)  ^—1  minute. 

d.  Counter-stain  with  &  weak  solution  of  eosin  until  sections  are  red. 

e.  Wash  in  water  until  pink. 

/.  Dehydrate  in  absolute  alcohol. 

g.  Clear  in  xylene  and  mount. 

Bacterium  diphtheriae.     Loeffler's  or  better  Weigert's  method. 

Bacillus  typhosus. 

a.  Loeffler's  methylen  blue  or  carbol-fuchsin  15  miu.-24  hrs. 

b.  Wash  slightly  in  distilled  water. 

c.  Place  in  10%  solution  of  tannic  acid  for  10-60  min. 

d.  Dehydrate  rapidly  in  alcohol. 

e.  Clear  in  xylene. 
/.  Examine. 

g.  Mount  in  balsam. 

Such  sections  examined  under  a  low  power  will  be  found  to  contain  heavily  stained 
masses,  which  under  a  high  power  prove  to  be  clumps  of  bacilli.     Not  infrequently  the 
bacilli  are  difficult  to  detect  in  tissue  from  typhoid  cadavers. 
Bacillus  suipestifer.     Loeffler's  method. 
Bacterium  Welchii.     Weigert's  and  Loeffler's  methods. 
Bacillus  chauvaei.     Use  Pfeiffler's  stain: 
a.  Dilute  carbol-fuchsin  J4  hour. 

1).  Absolute  alcohol  slightly  acidutated  with  acetic  acid  until  section  is  a  reddish 
violet  tint. 

c.  Xylene  and  examine. 

d.  Mount  in  balsam. 
Bacillus  oedematis.     Pfeiffer's  stain. 
Streptothrix  actinomyces. 

a.  Ziehl's  carbol-fuchsin,  10  minutes. 

b.  Wash  in  distilled  water. 

c.  Picri^  acid  (cons.  ale.  solution). 

d.  Wash  in  distilled  water. 

e.  Wash  in  alcohol  (50%). 

/.  Dehydrate  in  absolute  alcohol. 
g.  Clear  in  xylene. 
h.  Balsam. 

Tissue  stained  yellow,  rays  red. 
REFERENCES.     M.  &  W.  239-286;  N.  537. 


CHAPTER  IX. 

BACTERIOLOGICAL  DIAGNOSIS. 


EXERCISE  CVI.     EXAMINATION  OF  BUCCAL  SECRETION. 

DEFINITION.  The  secretion  of  the  mouth,  or  saliva,  is  a  mixed  product  derived  iu 
part  from  the  mucous  glands  within  the  mouth  and  also  from  the  parotid,  submax- 
illary,  and  sublingual  glands  In  disease  the  normal  character  of  the  different  parts  may 
vary  or  there  may  be  various  exudates  and  growths  present. 

COLLECTION.  Material  for  bacteriological  examination  is  best  obtained  by  means  of 
a  sterile  probang  or  forceps.  This  material  may  be  examined  directly  by  means  of 
cover-glass  preparations  or  by  means  of  cultures. 

1.  Method  of  Preparing  Outfit.     Wind  a  small  piece  of  absorbent  cotton  on  the  end 
of  a  wire  (about  1  mm.  in  diameter  and    14  cm.   long).     Thrust  the   other  end  of  the 
wire  through  the  cotton  plug  of  a  test-tube  or  fasten  in  a  cork  and  sterilize  at  150°  C.  for 
1  hour.     This  with  a  tube  of  nutrient  medium  (usually  Leoffler's  Blood  serum)  is  placed 
in  a  box  for  transportation. 

2.  Method  of  Using  Outfit.     The  patient  is  placed  in  a  good  light  and  the  probang 
gently  but  firmly  rubbed  over  the  suspected  area  of  the  throat  and  then  drawn  gently 
over  the  surface  of  the  medium,  both  tubes  securely  stoppered  and  the  outfit  sent  to  the 
laboratory.     The  organisms  to  be  sought  for  are  B.  diphtheriae,  the  pyogenic  cocci  and 
Monilia  Candida. 

BACTERIUM  DIPHTHERIAE. 

The  presence  of  this  germ  in  the  mouth  usually  results  in  a  formation  of  a  pseudo- 
membrane  a  portion  of  which  is  to  be  removed  with  a  pair  of  forceps  or  by  means  of  the 
outfit  described  above.  It  should,  1)  be  examined  directly  for  the  diphtheria  bacillus  by 
smearing  on  a  cover-glass  and  staining  by  following  methods : 

a.  Loeffler's  methylen  blue. 

6.  Gram's  stain. 

c.  Neisser's  stain:  a.  1  gram  methylen  blue  dissolved  in  20  cc.  of  alcohol  (96%),  is 
added  to  950  cc.  of  distilled  water  and  50  cc.  of  glacial  acetic  acid;  6.  2  grams  of  bismark 
brown  dissolved  in  a  liter  of  distilled  water.  Films  are  stained  in  a.  2  to  3  seconds,  washed 
in  water,  stained  in  6.  3  to  5  seconds,  dried  and  mounted. 

2)  Usually,  however,  mere  microscopical  examination  is  not  sufficient,  and  culture 
methods  must  be  employed.  In  fact  this  method  ought  always  to  be  used. 

In  this  case  make  smears  on  Loeffler's  blood  serum  and  incubate  them  at  36-38°  C. 
for  12-24  hours  and  then  examine  the  growth  in  cover-glass  preparations.  The  diphthe- 
ria organism  if  present  should  show: 

a.  Characteristic  appearance  with  Loeffler's  methylen  blue. 

b.  Positive  Neisser  stain. 

c.  Positive  Gram  stain. 


180 


Medical  Bacteriology. 


3)  Occasionally  micro-organisms  (pseudo-diphtheria  bacilli  among  others)  are  met 
with  that  very  closely  resemble  the  Klebs-Loeffler  bacillus  and  render  a  positive  diag- 
nosis doubtful.  In  such  cases  attention  to  following  table  will  be  helpful: 


1)  Form 


2)  Size 

3)  Threads 

4)  Grouping 

5)  Involution  forms 

6)  Motility 

7)  Stains 

a.  Loeffler's  methylen  blue 

6.  Gram 
c.  Neisser 

8)  Spores 


9)  Alkaline  potato 

10)  Sugar   agar    and     gelatin 

stab  cultures 

11)  Neutral  litmus  milk 

12)  Anaerobic  cultures  in  H 

13)  Nitroso-indol  reaction 

14)  Inoculation  experiments 
(Guinea  pig  subcutaneous) 


B.  Diphtheriae 


Slender  and  of  same  diameter 
throughout 

Average  1.2-2  M 

Not  formed 

Parallel  grouping  more  or  less 

characteristic  but  do  not  touch 

Common 

Immotile 

Stains  readily  giving   banded 

or  polar  stain 

Positive 

Characteristic  stain  with  very 

young  cultures,  six  hours. 

Absent 


Growth  almost  invisible 


Full  length  of  stab 
Acid  reaction 
Grows  well 
After  7  days 

Death  36-48  hours. 


B.  pseudo-diphtheriae 


Thicker  at  center  than  ends, 
plumper  and  shorter  ami  less 
variable  than  B.  diphtheriae 
Averaging  1-1.6  /* 

Not  formed 

Parallel  but  lie  closer  together 

Rare 
Immotile 

Stains  more  regularly 

Polar  stain  rare 

Positive 

Not  under  24  hours 

Absent 


Visible  and  cream  colored  in  2 
days 

Only  at  upper  part 
Alkaline  reaction 
No  growth 
After  21  days 

Non-pathogenic 


PYOGENIC  MICROCOCCI. 

1)  Stained  cover-glass  preparations  are  examined  and  if  micrococci  are  found  make: 

2)  Smear  cultures,  or  better  agar  plate  cultures  and  work  up  the  colonies  as  they 
appear. 

MONILIA  CANDIDA  (Organism  of  Thrush). 

The  material  is  collected  by  removing  a  portion  of  the  patches  or  membrane  and  ex- 
amining it: 

1)  Under  the  microscope  in  a  drop  of  glycerine. 

2)  Cover-glass  preparations  stained  with  carbol-fuchsin  or  Gram's  method. 

3)  By  means  of  smear  cultures  on  agar  or  blood  serum,  the  resulting  growth  being 
examined  either  in  glycerine  mounts  or  stained  cover-glass  preparations. 

REFERENCES,     v.  J.  95;   S.  101.     See  also  various  texts  under  special  organism. 

EXERCISE  CVH.      EXAMINATION  OF  SPUTUM. 

Definition.  By  this  term  is  meant  all  of  the  material  derived  from  the  air  passages 
by  the  act  of  coughing  or  hawking. 

METHOD  OF  COLLECTION.  For  diagnostic  purposes  it  is  best  collected  in  a  salt- 
mouthed  bottle  (about  2  oz.  capacity)  which  has  been  sterilized.  The  morning  sputum 
is  best  and  before  being  collected  the  mouth  should  be  rinsed  out  with  water. 


182  Medical  Bacteriology. 

BACTERIUM  TUBERCULOSIS.  Place  the  sputum  in  a  Petri  dish  over  a  black  surface 
and  select  one  of  the  little  cheesy  masses,  if  these  are  present,  and  smear  it  on  a  cover- 
glass.  Where  these  particles  are  not  present  a  loop  or  two  of  the  thick  portion  is  used. 
The  cover-glass  preparations  are  to  be  stained  by  one  of  the  following  methods: 

1)  Gabbett,  see  Part  1,  p.  38. 

2)  Ziehl-Neelson: 

<t.  Carbol-fuchsin  ten  times  through  the  flame- 

b.  Nitric  acid  (30%)  momentarily. 

c.  Water. 

d.  Alcohol  (60%)  until  red    color  disappears.     It  may  be  necessary  to  immerse 
preparation  in  acid  a  second  time,    but  the  greatest  care  must  be  exercised  to  prevent 
extraction  of  dye  from  tubercle  bacterium. 

e.  Loeffler's  methylen  blue,  1  minute. 
/.   Mount  and  examine. 

While  the  tubercle  bacteria  may  be  detected  when  present  in  considerable  numbers 
with  a  •$•  in.  objective  when  there  are  few  present  a  yV  in-  oil  immersion  will  be  neces- 
sary, and  this  ought  to  be  used  to  search  all  slides  where  the  tubercle  germ  has  not  been 
found  with  a  lower  power.  A  mechanical  stage  is  a  great  convenience  in  a  systematic 
search. 

At  least  two  preparations  should  be  stained  and  thoroughly  examined  before  a  neg- 
ative result  is  pronounced. 

The  viscosity  of  sputa  may  be  overcome  and  the  bacteria  concentrated  where  the  num- 
ber is  very  small  by  1)  Ribbert's  method  which  consists  in  the  addition  of  a  2%  solution 
of  caustic  potash  and  boiling.  This  dissolves  the  mucus  and  the  bacteria  are  then  depos- 
ited with  the  sediment.  This  sediment  can  be  obtained  by  allowing  the  mixture  to 
stand  in  a  conical  glass  vessel  or  more  quickly  by  the  use  of  a  centrifuge.  2)  Ham- 
mond's method: 

1.  Add  5%  of  crystallized  carbolic  acid  (in  the  case  of  sputum  add  5  times  its  bulk 
of  a  5  %  solution  of  carbolic  acid) . 

2.  Place  15  cc.  in  the  tubes  of  a  centrifuge  and  whirl  for  15  minutes. 

3.  Pour  off  supernatant  fluid  and  treat  precipitate  with  3  cc.  of  a  5%  KOH  solution. 
Mix  thoroughly  and  allow  to  stand  2  minutes. 

4.  Fill  to  15  cc.  mark  with  distilled  water  and  whirl  20  minutes. 

5.  Make  cover-glass  preparation  of  sediment  (or  purify  same  by  repeated  washings 
and  centrifugalizations  with  distilled  water) . 

A  centrifugal  machine  should  be  able  to  make  at  least  2,500  revolutions  per  minute- 
This  speed  ought  to  be  maintained  for  15  minutes.  Sputum  may  be  preserved  by  ad- 
dition of  small  quantity  of  carbolic  acid  (5%). 

Negative  results  are  of  positive  diagnostic  value  only  when  repeated  examinations 
are  made  of  different  samples  taken  at  different  times. 

BACTERIUM  INFLUENZAS  .  This  micro-organism  is  frequently  present  in  enormous 
numbers  (100  or  more)  and  sometimes  in  almost  pure  cultures  in  the  greenish  purulent 
masses  in  the  sputum.  It  stains  readily  with  the  ordinary  dyes,  and  when  lightly  stained 
presents  the  bipolar  stain.  Carbol-fuchsin  diluted  10  times  is  one  of  the  best  stains. 
Gram's  stain  is  negative. 

Sputum  from  suspected  cases  should  be  collected  either  by  means  of  a  probang  or 
in  a  bottle  and  examined : 


184  Medical  Bacteriology. 

1)  Microscopically  by  staining,  with  a  weak  carbol-fuchsin,  smears  from  the  puru- 
lent masses.     If  a  very  small  bacillus  is  in  large  clumps,  which  fails  to  retain  stain  by 
Gram's  method,  the  evidence  is  strong  that  it  is  the  influenza  bacillus;   the  diagnosis 
should  be  confirmed,  however,  by 

2)  Cultures  on  blood  agar. 

Animal  inoculations  are  without  effect. 

BACTERIUM  PNEUMONIAE. 

The  sputum  of  patients  suffering  from  pneumonia  is  usually  of  a  rusty  color  due  to 
presence  of  blood.  The  "pneumococcus"  is  readily  seen  in  such  material  when  stained 
by  Gram's  method,  or  with  carbol-fuchsin  and  momentarily  washed  with  alcohol,  as 
lancet-shaped  organisms  with  outer  ends  pointed  and  surrounded  by  a  clear  area — the 
capsule.  The  capsule  can  be  easily  stained  by  Welch's  method.  (See  XXXVI.) 

This  organism  is  also  frequently  found  in  the  sputum  of  healthy  persons  and  small 
numbers  may  be  detected  by  means  of  animal  inoculation.  The  rabbit  or  mouse  are 
most  susceptible  and  should  be  inoculated  intraperitoneally.  As  a  result  of  infection 
with  this  organism  the  animal  quickly  dies  with  a  typical  septicaemia,  the  micro-organ- 
isms being  found  in  great  numbers  in  the  blood  current. 

BACILLUS  PESTIS.  This  micro-organism  is  frequently  found  in  the  sputum  especially 
in  the  pneumonic  form  of  the  disease — for  methods  of  detection  see  CX. 

STREPTOTHRIX  ACTINOMYCOSES.     This  organism  has   been   occasionally   found   in 
sputum  and  in  such  cases  the  peculiar  morphology  of  the  colonies  is  well  brought  out 
by  Gram's  method.     See  CX. 
REFERENCES,     v.  J.  114;   S.  245.     See  also  various  texts  under  particular  organisms. 

EXERCISE  CV1II.     EXAMINATION  OF  BLOOD. 

For  serum  test  (Widal  reaction)  the  blood  may  be  collected  and  dried  (see  below), 
but  in  other  cases  where  cultures  are  to  be  made  the  blood  must  be  collected  aseptically 
in  sterile  receptacles  and  hermetically  sealed-  For  this  purpose  Sternberg's  bulb  is  ex- 
cellent. The  skin  should  first  be  sterilized  by  use  of  corrosive  sublimate  or  carbolic  acid 
followed  with  alcohol. 

It  is  usually  well  in  any  case  to  make  cover-glass  smears  at  the  bed-side  for  micro- 
copical  examination.  These  are  best  made  as  follows:  Place  a  drop  of  blood  about 
the  size  of  a  pin-head  on  a  perfectly  clean  cover-glass  and  then  a  second  cover-glass  on 
this;  this  flattens  the  drop  of  blood  out  into  a  thin  film.  Immediately  and  before  coag- 
sulation  can  take  place  the  two  are  drawn  apart  horizontally  and  the  films  allowed  to  dry. 
(Cabot.) 

BACTERIUM  ANTHRACIS.  In  case  of  animals  dead  of  suspected  anthrax,  blood  or 
portion  of  spleen  should  be  removed  with  least  possible  danger  from  infection  or  distri- 
bution of  bacilli  and  studied  as  follows: 

1.  Microscopical  examinations  of  blood  or  the  spleen  pulp  of  animals  show  (when 
stained  with  Loeffler's  methylen  blue)  large  bacteria  in  chains  (5  or  6  segments)   pre- 
senting the  bamboo  appearance. 

2.  In  hanging  drop  preparation  large,  homogeneous,  immotile  bacilli. 

3.  Agar  plate  cultures  should  also  be  made  and  from  the  separate  colonies  subcul- 
tures;  the  gelatin  stab  being  especially  characteristic. 


186  Medical  Bacteriology. 

4.  In  important  cases  (as  in  man)  guinea  pigs,  or  white  mice,  should  be  inoculated, 
and  in  case  of  death  organism  isolated  and  identified. 

SPIRILLUM  OBERMEIERI.  This  organism  is  found  in  the  blood  only  during  a  par- 
oxysm. It  is  a  long  slender  orgaiiism  6  or  7  times  the  diameter  of  a  red  blood  corpuscle. 
(45/J-)  They  have  a  brisk  vibratile  movement  in  the  direction  of  their  long  axis.  They 
are  very  sensitive  to  reagents  of  all  kinds.  Even  the  addition  of  distilled  water  will  cause 
them  to  disappear.  Fresh  blood  is  best,  but  dried  smears  may  be  used  and  stained 
with  fuchsiu  or  by  Gunther's  method: 

a.  Dried  films  are  treated  with  acetic  acid  (5%)    10  seconds,  this  is  removed  by 
blowing  and  holding  film  over  flask  of  strong  ammonia  previously  shaken. 

b.  Stained  in  Ehrlich's  gentian  violet. 

c.  Washed  with  water. 

d.  Dried. 

e.  Mounted  in  balsam  or  xylene. 
/.  Examined. 

PYOGENIC  MICROCOCCI.  These  are  occasionally  found  and  for  method  of  detection 
see  CX. 

BACTERIUM  MALLEI.  Sometimes  found  in  the  blood  of  those  suffering  with  Gland- 
ers. It  may  be  detected  in  the  blood-smears.  For  special  methods  see  CX. 

B.  PNEUMONIAE.  This  germ  is  frequently  present  in  fatal  cases  24  to  48  hours  be- 
fore death.  The  blood  should  be  drawn  with  a  sterile  hypodermic  syringe  and  about 
1  cc.  of  blood  mixed  with  a  tube  of  melted  a  gar  at  43°C.  and  poured  into  a  Petri  dish. 
Characteristic  colonies  appear  in  24  to  48  hours. 

B.  TUBERCULOSIS.  In  case  of  miliary  tuberculosis  they  may  be  very  rarely  found  in 
sufficient  numbers  to  be  detected  by  staining  methods,  see  sputum  CVII. 

B.  INFLUENZAE.  Canon  claims  to  have  stained  and  cultivated  this  organism  in  blood, 
but  this  needs  confirmation. 

B.  COLI.  This  organism  may  be  found  in  the  blood,  for  methods  of  isolation  and 
identification  see  Faeces  CIX. 

BACILLUS  PESTIS.  This  germ  occurs  in  the  blood  in  certain  cases  at  least  but  ap- 
pears to  require  considerable  skill  in  detecting  it  due  to  its  variable  appearance.  Broth 
tubes  should  be  infected  and  animals  inoculated. 

BACILLUS  SUIPESTIFER. 

a.  Make  agar  plate  and  streak  cultures  from  spleen  of  dead  animal,   and  work  up 
the  colonies  as  they  appear. 

b.  Widal  Reaction  (for  technique  see  below  under  B.  typhosus). 
PLASMODIUM  MALARIAE. 

a.  Examination  of  fresh  blood.     A  droplet  of   blood  from  finger  or  lobe  of  ear  is 
placed  on  a  glass  slide,  covered  with  a  cover- glass  and  then  the  cover-glass  is  ringed  with 
vaselin.     Examination  should  be  made  with  a  yV  in-  oil  immersion. 

b.  Stained.      Prepare  films  as  directed  above  and  stain  with  methylen  blue   and 
eosin  or  treat  films  with  a  very  weak  acetic  acid  2  or  3    drops  to  30  cc.  of  water;   to 
remove  haemoglobin  wash  with  water  and  stain  with  following  solution  for  ^  minute : 
Borax  5.0  parts. 
Methylen  blue  0.5  parts. 
Water                                                                                                                         100.0  parts. 

Wash,  dry  and  mount  in  balsam  (Manson). 


188  Medical  Bacteriology. 

BACILLUS  ICTEROIDES.  Make  agar  streaks  from  blood  or  fragment  of  liver  (where  liver  is 
obtained  it  is  best  wrapped  in  cloth  and  kept  in  incubator  at  38°C.  for  12  hours  before 
cultures  are  made  to  encourage  development  of  the  micro-organisms,  which  are  usually 
only  sparingly  present  in  tissue).  Keep  the  cultures  at  38°C.  for  12-16  hours  and 
then  at  22°C.  for  same  time;  the  characteristic  appearance  is  a  transparent,  bluish 
growth  surrounded  by  an  opaque  zone.  If  this  is  not  obtained  other  cultures  must  be 
prepared  and  a  thorough  study  of  the  organisms  isolated  made. 

REFERENCES,     v.  J.  45;   S-  79.     See  also  texts  under  particular  organisms. 

WIDAL  REACTION.  Directions  for  collecting  samples  of  blood.  "  Wash  with  boiled 
water  the  part  from  which  the  blood  is  to  be  obtained  (lobe  of  ear,  end  of  finger,  or  toe 
in  infant).  Prick  deeply  the  skin  with  a  clean  needle."  Remove  two  or  three  large  drops 
of  blood  on  a  clean  glass  slide,  alluminum  foil,  piece  of  isinglass  or  letter  paper. 

Allow  the  blood  to  dry.  Then  place  in  an  envelope  and  send  to  laboratory  and  test 
as  follows : 

a.  Make  a  hanging  drop  preparation  from  a  24-72  hour  old  agar,  or  bouillon,  cul- 
ture of  Bacillus  typhosus. 

b.  If  the  bacilli  are  actively  motile,  remove  the  cover-glass,  add  to  the  culture  a 
small  drop  of  a  solution  of  typhoid  blood  (diluted  from  10-50  times),  return  the  cover- 
glass  to  the  slide  and  seal  well  with  vaselin. 

c.  Examine  with  a  high  dry  power  (|  in.  obj.)  rather  than  with  the  oil  immersion. 
In   a    typical  reaction  the  motility  is  almost  immediately  affected  and  soon  motion 

ceases  altogether  while  the  bacilli  collect  in  clumps,  i.  e.  become   "agglutinated." 
REFERENCES,     v.  J.  45;   S.  79.     See  also  texts  under  particular  organism. 

EXERCISE  CIX.  EXAMINATION  OF  FAECES. 

The  material  expelled  from  the  rectum  and  comprising  the  substances  from  the 
food  and  the  secretions  of  the  alimentary  tract  come  under  this  head.  The  number  of 
micro-organisms  occuring  here  is  enormous,  and  comprise  a  large  number  of  species  and 
among  them  several  pathogenic  forms  particularly  B.  typhosus.  M.  comma,  B.  tubercu- 
losis and  Amoeba  coli. 

BACILLUS  TYPHOSUS.  This  organism  occurs  in  the  faeces  in  the  case  of  typhoid  pa- 
tients, but  on  account  of  the  large  number  of  other  organisms  its  detection  is  very  diffi- 
cult The  following  methods  are  the  most  serviceable : 

Parietti's  Method.  This  method  consists  in  adding  Parietti's  solution  (carbolic 
acid  5  grams:  hydrochloric  acid  4  grams,  and  distilled  water  100  cc.)  to  bouillon  in 
the  following  manner:  A  number  of  tubes  of  bouillon  have  a  varying  quantity  of  the 
above  solution  added,  e.  g,  1  drop  to  one  tube,  2  to  another,  3  to  another,  and  so  on. 
These  tubes  are  inoculated  with  a  small  quantity,  (one  or  two  loops),  of  the  faeces 
and  then  placed  in  the  38°  C-  incubator.  Twenty-four  hours  later  the  tube  containing 
the  largest  amount  of  Parietti's  solution  which  shows  growth  probably  contains  B.  coli 
and  B.  typhosus  if  it  is  present.  The  organisms  may  be  separated  most  quickly  and 
easily  by  the  use  of  the  lactose  litmus  agar  plate.  The  blue  colonies  should  be  worked 
up,  and  especially  tested  for  its  agglutinating  power  on  typhoid  blood.  Instead  of  the 
use  of  the  lactose  litmus  agar  plate,  either  Eisner's  or  Hiss'  methods  may  be  used. 


190  Medical  Bacteriology. 

Eisner's  Medium.     Method  of  preparation: 

Peel  and  cut  up  500  gms.  of  old  potatoes  of  medium  size,  add  1000  ce.  of  water  and 
boil  1  and  £  hours. 

Mash  potatoes  thoroughly ;  strain  through  a  cloth  and  add  water  to  filtrate  to  make 
a  liter. 

Add  15  %  gelatin  and  boil  10  minutes.  Cool  to  60°  C.  and  add  white  of  one  egg 
and  boil  15  minutes. 

Filter  through  cotton,  then  paper.  Titrate  and  make  gelatin  2-3  %  acid.  Just  before 
tubing  add  1  %  potassium  iodide  (10  cc.  of  a  solution  in  which  1  cc.  contains  1  gram  of 
potassium  iodide).  Tube  and  sterilize  three  times. 

Plates  of  this  medium  are  made  in  the  usual  way  and  kept  at  15-18°  C.  On  this  me- 
dium the  typhoid  germ  forms  very  finely  granular,  small,  bright  droplets  resembling 
condensed  moisture,  while  the  colon  bacillus  gives  rise  to  larger,  brown  colonies,  which 
are  more  granular  and  spread  more. 

Hiss'  Plate  Medium.     This  contains- 
10  grams  of  agar. 
25  grams  of  gelatin. 
5  grams  of  beef  extract  (Leibig). 
5  grams  of  sodium  chloride. 
10  grams  of  glucose. 
1000  grams  of  water. 

It  is  made  by  first  dissolving  the  agar,  salt  and  extract  in  the  water,  then  the  gelatin 
is  added  and  dissolved,  the  reaction  changed  by  use  of  NaOH  and  phenolphthalein  so 
that  it  will  contain  not  less  than  2%  normal  acid,  cleared  with  two  eggs  and  filtered,  glu- 
cose added  and  the  medium  tubed  and  sterilized. 

Make  plate  cultures  in  ordinary  way  and  incubate  at  38°  C.  for  18  hours,  then  ex- 
amine the  colonies  microscopically.  The  colonies  of  B.  typhosus  have  irregular  out- 
growths and  fringing  threads.  The  colonies  of  B.  coli,  on  the  other  hand,  are  much  large 
and  as  a  rule  are  darker  in  color  and  do  not  form  threads. 

The  colonies  may  be  further  examined  by  the  use  of  Hiss'  Tube  Medium. 

5  grams  of  agar-agar. 
80  grams  of  gelatin. 
5  grams  beef  extract  (Leibig) . 
5  grams  sodium  chloride. 
10  grams  glucose. 
1000  grams  water. 
Made  as  plate  medium  except  that  is  is  to  contain  1.5%  normal  acid. 

Within  18  hours  at  38°  C.  the  typhoid  bacilli  produce  a  uniform  clouding.  The 
colon  bacilli  do  not  produce  uniform  clouding  and  do  produce  gas- 

All  suspected  cultures  should  be  tested  with  typhoid  blood  (Widal  reaction). 
The  typhoid  organism  may  be  isolated  from  the  stools  during  the  first  two  weeks  of 
the  disease. 

MlCEOSPIRA  COMMA. 

1.  Microscopical  examination  of  "rice-water"  discharges  for  spirilla  lying  parallel. 

2.  Culture  methods.     Gelatin  or  agar-plates  should  be  made  from  the  rice-like  flakes; 
other  flakes  should  be  inoculated  into  flasks  of  peptone  water  (Dunham's  solution)  and 
inoculated  at  38°  C.     The  surface  growth  6-12  hours  later  is  to  be  examined  microscop- 


192  Medical  Bacteriology. 

ically  and  by  means  of  plates.  Then  test  the  peptone  cultures  for  nitroso-indol  (cholera 
red  reaction)  by  the  addition  of  a  few  drops  of  sulphuric  acid. 

BACTERIUM  TUBERCULOSIS.  This  organism  has  been  found  in  the  stools  in  cases 
of  intestinal  ulcerations,  and  may  come,  in  cases  of  phthisis,  from  ingested  sputa. 

AMOEBA  COLI. 

1.  A  drop  of  the  mucous  portions  of  stool  is  placed  on  a  glass  slide,  covered  with  a 
cover-glass  and  examined  with  a  magnification  of  about  500  diameters  (-5-  in-  objective). 
Examination  should  be  conducted  on  a  warm  stage  in  order  to  get  amoeboid  movements. 

2.  Preparations   may  be   stained  with  methylen  blue   and  carmine.     The  nucleus 
is  stained  with  the  carmine. 

3.  Discharge  may  be  hardened  and  stained  by  Mallory's  method  as  follows: 

a.  Fix  tissues  in  alcohol. 

b.  Stain  (paraffin)  sections   in   a  saturated  aqueous    solution  of   thionin  for  5-20 
minutes. 

c.  Wash  in  water. 

d.  Differentiate  in  a  2%  aqueous  solution  of  oxalic  acid  }4—l  minute. 

e.  Wash  in  water. 

/.   Dehydrate  in  alcohol  (95%). 

g.  Clear  in  oil  of  bergamot. 

h.  Wash  with  xylene  and  mount  in  balsam. 

Nuclei  of  Amoebae  brownish  red,  other  nuclei  blue. 

REFERENCES,     v.  J.  199;   Si.  228.     See  also  texts  under  various  organisms. 

EXERCISE  CX.     EXAMINATION    OF  URINE. 

For  bacterial  examination  urine  should  be  drawn  with  a  sterile  catheter  into  a  sterile 
bottle. 

BACTERIUM  TUBERCULOSIS. 

For  method  of  staining  see  under  Sputum,  CVII. 

It  is  best  to  centrifuge  the  product  and  care  must  be  taken  to  differentiate  from  the 
Smegma  bacterium.  For  this  purpose  stain  cover- glass  smears  as  follows  (Bunge  & 
Franteroth.): 

1)  Absolute  alcohol,  3  hours. 

2)  Chromic  acid,  15  minutes. 

3)  Stain  in  hot  carbol-fuchsin. 

4)  Decolorize  in  sulphuric  acid  (25%)  2-3  minutes. 

5)  Counter-stain  with  a  saturated  alcoholic  solution  of  methylen  blue. 
The  smegma  bacillus  is  decolorized  by  this  method. 

Tubercle  bacterium  in  urine  is  frequently  present  in  clusters  while  the  smegma 
bacterium  occurs  singly.  Injection  of  guinea  pigs,  smegma  bacillus  is  non-pathogenic. 

The  following  organisms  have  also  been  found  in  the  urine.  For  methods  of  isolation 
see  references. 

PYROGENIC  MICROCOCCI.     CXI. 

M.  GONORRHOEAE.      CXI. 

B.  TYPHOSUS.      CIX. 

S.  OBERMEIERI.       CVIII. 

REFERENCES,     v.  J.  273;   Si.  504.  and  texts  under  the  various  organisms. 


194  Medical  Bacteriology. 

EXERCISE  CXI.     EXAMINATION  OF  TRANSUDATES  AND  EXUDATES. 

The  material  should  be  collected  in  sterile  vessels  under  aseptic  precautions.  Make 
several  cover-glass  preparations  and  stain  one  with  Loeffler's  methylen  blue  and  the 
others  with  gentian  violet  or  carbol-fuchsin.  Mount  and  examine. 

a.  If   staphylococci  alone  are  present  search  for  the  pyogenic  micrococci. 

b.  If  streptococci  suspect  S.  pyogenes. 

c.  If  diplococci  or  tetracocci. 

1.  Within  the  pus-cells  test  for  M.  gonorrhoeae  or  M.  intracellularis. 

2.  Free.  8.  tetragena. 

d.  If  bacilli  any  of  the  following  may  be  searched  for: 

1.  B.  colt.  This  organism  is  likely  to  be  found  especially  in  suppurative 
peritonitis  and  diseases  of  the  urinary  organs.  2.  B.  anthracis.  3.  B.  pneumoniae. 
4.  B.  tuberculosis.  5.  B.  leprae.  6.  B.  mallei.  7.  B.  pestis.  8.  P.  aeruginosa.  9. 
B.  welchii.  10.  B.  oedematis.  11.  B.  tetani. 

e.  Streptothrix  actinomyces. 

f.  Amoeba  coli. 

PYOGENIC  MICROCOCCI.  These  organisms  are  frequ  ently  present  in  pus  and  should  be 
isolated  and  identified  in  pure  cultures  as  microscopical  examinations  alone  will  not, 
suffice. 

STREPTOCOCCUS  PYOGENES.  This  organism  is  not  infrequently  present  and  can  be 
readily  identified  by  culture  methods. 

MICROCOCCUS  GONORRHOEAE.     Pus  should  be   collected  in  a  sterile   receptacle   or 
spread  on  cover-glasses  and  allowed  to  dry,  but  should  not  be  allowed  to  dry  and  then  wet 
up  again  to  spread,  as  this  destroys  the  pus-cells,  and  hence  the  value  of  the  material  for 
diagnosis. 
Stain: 

1.  a.  Loeffler's  methylen  blue  3-5  minutes. 

b.  Wash  in  water. 

c.  Dry,  mount  in  balsam  and  examine  with  jV  in.  oil  immersion. 

d.  Look  for  a  biscuit- shaped  diplococcus  within  the  pus-cells. 

2.  By  Gram's  method- 

a.  Anilin  oil  gentian  violet  15  minutes. 

b.  Wash  in  water- 

c.  Treat  with  iodine  solution  2  minutes. 

d.  Decolorize  with  alcohol. 

e.  Counter- stain  with  Bismark  brown,  f  minutes. 
/.    Wash,  dry  and  moiiut  in  balsam. 

g.  Examine  with  oil  immersion. 

If  the  gonococci  are  present  they  will  be  stained  brown. 
If  diagnosis  is  of  great  importance  make  cultures  as  follows: 

1)  Make  6  or  more  streak  cultures  on  blood  agar  or  better  make  plates  on  Wertheim's 
medium  (p.  99).     Grow  at  38°  C. 

2)  Make  a  set  of  ordinary  agar  plates  or  streak  cultures  and  keep  at  38°  C. 

The  gonococcus  grows  on  the  first  two  media  but  not  on  the  plain  agar.  The 
gonococcus  is  the  only  organism  that: 


196  Medical  Bacteriology. 

1)  Occurs  in  groups  (cell-colonies)  in  pus-cells. 

2)  Is  decolorized  by  Gram's  method. 

3)  Does  not  grow  on  agar  at  room  or  blood  heat-     (Foulerton). 

MlCROCOCCUS  INTRACELLULARIS. 

Pus  may  be  obtained  by  lumbar  puncture  which  is  performed  as  follows.  The  back 
of  the  patient  and  the  operator's  hands  should  be  made  sterile.  The  needle  (4cm.  x  1 
mm.  for  children)  should  be  boiled  10  minutes.  The  patient  should  lie  on  the  right  side, 
with  the  knees  drawn  up  and  the  uppermost  shoulder  so  depressed  as  to  present  the  spinal 
column  to  the  operator.  The  puncture  is  generally  made  between  the  third  and  fourth 
lumbar  vertebrae.  The  thumb  of  the  left  hand  is  pressed  between  the  spinous  processes 
and  the  point  of  the  needle  is  entered  about  1  cm.  to  the  right  of  the  median  line, 
and  on  a  level  with  the  thumb  nail  and  directed  slightly  upwards  and  inward  toward  the 
median  line.  At  a  depth  of  3  or  4  cm.  in  children  and  7  or  8  in  adults  the  needle  enters  the 
subarachnoid  space  and  the  fluid  flows  usually  by  drops.  This  is  allowed  to  drop  into  an 
absolutely  clean  test-tube,  which  has  previously  been  plugged  and  sterilized.  From  5  to  15 
cc.  of  the  fluid  is  a  sufficient  quantity  for  examination.  Cultures  should  be  made  at  once  on 
blood  agar  and  plain  agar  (M.  &  W.  371.).  After  standing  some  hours,  the  sediment 
should  be  examined  in  cover-glass  preparations,  stained  with  Loeffler's  methylen  blue 
and  by  Gram's  method. 

Micrococcus  intracellularis  stains  by  Loeffler.'s  method  and  appears  as  a  diplococcus 
in  groups  in  the  pus  cells,  is  decolorized  by  Gram's  method,  and  grows  on  blood-agar 
and  feebly  on  ordinary  agar  at  38°  C. 

The  following  organisms  are  also  found  occasionally.  For  methods  of  diagnosis  see 
exercises  indicated. 

B.  COLI.     CIX. 

B.  PNEUMONIAE.     Stain  for  capsule.     Cultivate  on  blood-agar.     CVII. 

B.    TUBERCULOSIS-       CVII. 

B.  LEPRAE.     For  method  of  staining,  see  CV. 

B.    MALLEI. 

a.  Widal  reaction  (If  in  man  typhoid  and  diphtheria  must  "be  excluded  in  case  of  a 
positive  reaction). 

b.  Examination  of  discharge. 

1.  Microscopical  examination  usually  without  result. 

2.  Cultures,  glycerine  agar  and  potato  from  pus. 

c.  Animal  inoculation,  Straus  method. 

B.    PESTIS. 

a.  Make  plate  cultures  from  blood  and  buboes  and  work  up  colonies. 

b.  Make  subcutaneous  inoculation  into  guinea  pigs  from  bubo,  and  if  death  ensues 
search  for  B.  pestis. 

P.  AERUGINOSA.     Easily  recognized  by  its  culture  characters. 

B.    WELCHII. 

This  germ  is  non-pathogenic  for  rabbits  but  Welch  and  Flexner  have  shown  that 
if  a  rabbit  is  inoculated  intravenously  with  0.5  to  1  cc.  of  a  bouillon  culture  and  killed 
after  a  lapse  of  5  or  10  minutes  and  the  animal  kept  at  18°-20°  C.  for  24  hours  or  at 
30°-35°  C.  for  4  to  6  hours,  the  organism  will  multiply  in  the  blood  and  produce  large 
quantities  of  gas  in  the  vessels  and  organs.  This  effect  is  characteristic. 


• 

*  * 


198  Medical  Bacteriology. 

B.    OEDEMATIS. 

a.  Make  cover- glass  preparations  from  fluid  of  affected  parts- 

b.  Also  make  anaerobic  cultures.     If  material  contains  spores  it  should  be  heated 
to  80°  0.  for  10  minutes  before  it  is  seeded- 

B.  TETANI. 

a.  Make  cover-glass  preparation  from  pus  and  search  for  drumstick  bacillus. 

ft.  Make  cultures  in  glucose  bouillon  and  agar-plates  and  develop  in  hydrogen. 

c.  Inoculate  animals   with  the  discharge,  and  also  with  the  bouillon  culture,  and 
watch  for  characteristic  symptoms. 

S.    ACTINOMYCES. 

a.  Place  one  of  the  minute  sulphur  yellow  nodules  in  a  drop  of  glycerine  on  a  glass 
slide  and  then  apply  gentle  pressure. 

b.  Even  the  low  powers  of  a  compound  microscope  will  then  show  something  of  the 
clustered  arrangement  which  can  be  more  carefully  studied  under  a  higher  power. 

c.  Intraperitoneal  inoculation  of  guinea  pig.  One  month  later  nodules  on  peritoneum. 
AMOEBA  COLI.     CIX. 

REFERENCES,  v.  J.  405;  Si.  514  and  518-  See  also  texts  under  the  various  or- 
ganisms. 

EXERCISE  CXII.     DIAGNOSIS  OF  RABIES. 

a.  The  medulla  of  the  suspected  animal  is  removed  under  aseptic  precautions,  as 
soon  as  possible  after  death.  In  case  the  animal  is  some  distance  from  the  laboratory  it 
is  best  to  cut  off  the  head,  pack  it  in  ice  and  ship  by  express. 

ft.  Place  a  piece  of  the  medulla  about  the  size  of  a  pea,  in  4  or  5  cc.  of  sterile  bouillon 
and  thoroughly  grind  UD  the  same. 

c.  Anaesthetize  a  rabbit  with   ether,  clip  the  hair  from  between  the  eyes  and  ears 
and  disinfect  with  a  carbolic  acid  solution. 

d.  Make  a  longitudinal   incision  through  the  skin  and  subcutaneous  tissue   along 
the  median  line,  while  a  crucial  incision  is  made  through  the  periosteum  on  one  side  of 
median  line  thus  avoiding  haemorrhage  from  the  longitudinal  sinus.     The   periosteum  is 
then  pushed  back  and  a  disc  of  the  skull  (|  inch  in    diameter)  removed  with  a  trephine 
and  the  dura  mater  exposed. 

e.  With  a  sterile  hypodermic  syringe  introduce   2  or  3  drops  of  the  suspension  of 
medulla  beneath  the  dura  mater,  stitch  the  skin,  disinfect,  dry  and  seal  the  wound  with 
collodion. 

The  rabbits  apparently  experience  no  inconvenience;  the  wound  heals  rapidly  and 
the  rabid  symptoms  appear  in  from  15  to  30  days,  although  sometimes  they  may  occur 
earlier  or  much  later. 

EXERCISE  CXIII.     EXAMINATION  OF  MATERIAL  FROM  HUriAN  AUTOPSIES. 

At  human  autopsies  smears  from  the  organs  should  be  made  on  cover-glasses  and 
afterwards  stained  and  examined.  Plate-cultures  should  also  be  made  from  the  various 
organs  or  instead  parallel  streaks  over  blood  serum,  agar-slopes  or  agar-plates.  In  all 
eases  the  surface  from  which  the  material  is  to  be  obtained  should  first  be  burned  to 
avoid  infection  of  cultures  with  extraneous  germs.  Portions  of  the  various  organs 
should  also  be  preserved  and  hardened  in  alcohol. 


CHAPTER  X. 

DETECTION  OF  PATHOGENIC  BACTERIA  IN  WATER  AND  MILK 

SUPPLIES. 


EXERCISE  CXIV.     EXAMINATION  OF  WATER  FOR  PATHOGENIC  BACTERIA. 

BACILLUS  TYPHOSUS.  In  the  examination  of  water  it  is  best  to  concentrate  the 
bacteria  by  filtering  a  large  amount  of  the  water  through  a  Berkefeld  filter  and  use  the 
slime  on  the  filter  to  make  the  plates. 

a.  Parietti's  method,  see  CIX. 

6.  Hiss'  method.  Make  plate  cultures  and  incubate  at  38°  C.  for  18  hours.  Inocu- 
late suspicious  colonies  into  Hiss'  tube  medium,  fermentation  tube,  milk  and  make  indol 
test.  Also  try  Widal  reaction. 

c.  Animal  Inoculation.     (Michigan  method). 

1)  Inoculate  suspected  water  into  bouillon  tubes  or  flasks,  and  incubate  at  38°  C. 

2)  Twenty-four  to  forty-eight  hours  later  inoculate  one  cc.  into  the  peritoneal  cavity 
of  a  white  rat. 

3)  If  animal  recovers  B.  typhosus  is  not  present.      If  animal  dies  hold  autopsy  and 
isolate  and  study  organism  causing  death. 

MICROSPIRA  COMMA. 

a.  If  there  is  reason  to  believe  that  the  spirilla  are  very  numerous  gelatin  plate  cul- 
tures can  be  made  directly  from  the  water,  and  the  suspicious  colonies  worked  up. 

l>.  Ordinarily  the  organisms  are  very  sparse  and  large  quantities  must  be  used,  100- 
1000  cc.  are  placed  in  flasks  aud  1%  of  peptone  and  0.5%  salt  are  added,  the  fluid  made 
alkaline  and  incubated  at  38°  C.  for  6-24  hours.  Then  gelatin  plate  cultures  are  made  from 
the  upper  layers  and  the  suspicious  colonies  worked  up  as  above. 

EXERCISE  CXV.     EXAHINATION  OF   HILK  FOR  PATHOGENIC  BACTERIA. 

B.  DIPHTHERIAE. 

Where  B.  diphtherias  is  suspected  in  milk,  make  a  considerable  number  of  streak 
cultures  on  Loefflers's  blood  serum  and  incubate  at  38°  C.  for  8-12  hours  and  examine 
growth  microscopically  very  carefully  for  B.  diphtheria*-. 

BACTERIUM  TUBERCULOSIS  (Koch)  Mig. 

Hammond's  method  of  examining  milk  for  B.  Tuberculosis.  See  Sputum,  CVII. 

Animal  Inoculation. 


Concerning  the  transmission  of  material  containing  Bacteria  in  Mails,  see  Postal  Guide,  1898 
Ruling  No.  82,  p.  901.  Part  of  which  is  as  follows:  "That  the  order  of  the  Postmaster  General  of 
June  1,  18!)li,  forbidding  the  use  of  mails  for  the  transmission  of  specimens  of  germs  of  cholera  or 
other  diseased  tissues,  is  hereby  modified  to  this  extent:  "Specimens  of  diseased  tissue  may  be 
admitted  to  the  mails  for  transmission  to  United  States,  State  or  municipal  laboratories  only  when 
inclosed  in  mailing  packages  constructed  in  accordance  with  the  specifications  hereinafter  enumer- 
ated. Upon  the  outside  of  every  package  shall  be  written  or  printed  the  words:  'Specimen  for 
Bacteriological  examination.'  No  package  containing  diseased  tissue  shall  be  delivered  to  any  rep- 
resentative until  a  permit  shall  have  first  been  issued  by  the  Postmaster  General,  certifying  that 
said  institution  has  been  found  to  l>e  entitled,  in  accordance  with  the  requirements  of  this  regula- 
tion, to  receive  such  specimens." 

[200] 


INDEX. 


ABBE    condenser.  22. 
Acids,  detection  of  in  cultures,  52; 
quantitative  determination  of,  52. 

Agar,  glucose,  44;  glycerine,  115; 
hanging-drop  culture  in,  30;  lac- 
tose, 44. 

Agar  plate  cultures,  34;  character  of 
colonies  on,  58. 

Agar  slopes,  12. 

Air,  analysis,  comparative,  80;  quan- 
titative, 80. 

Amoeba  coli,  in  faeces,  192;  in  exu- 
dates,  198. 

Ammonia,  detection  of  in  cultures,  52. 

Anaerobic  cultures,  149. 

Animal  inoculation.  162. 

Anilin  dyes,  18. 

Anilin  oil  gentian  violet,  18. 

Antiseptic  action,  48. 

Antiseptics,  method  of  testing,  84. 

Aspirator,  80. 

Autoclave,  8. 

Autopsies,  examination  of  material 
from,  198. 

BACILLUS  acidi  laclici,  70. 
aerogenes  capsulatus,  150, 
amylobacter,  40. 
of  anthrax,  104. 
campestris,  46. 
of  blue-green  pus,  130. 
of  bubonic  plague,  124. 
ckauvaei,  152;  172;  176. 
of  chicken  cholera,  110. 
coli,   16;  24;  84;  36;  46;   48;   52;    54; 

in  the   blood,  186;    in  transu- 

dates  and  exudates.  196. 
of  diphtheria,  118. 
of  Friedlander,  108. 
of  glanders,  116. 
of  hog  cholera,  128. 
icteroides,  128;  188. 
of  influenza,  120. 
of  malignant  oedema,  154. 
mycoidts,  26 

oedematis,  154;  172;  176;  198. 
pestis,  124;  164;  184;  186;  196. 
prodigiosus,  50;  56;  64;  67. 
pyocyaneus,  130. 
rouget  du  pore,  112. 
of  septicaemia  haemorrhagica,  110. 
subtilis,  16;  22;  24;  30;  84;  SB;  40;  46; 

48;  50;  52;  54. 
suiptstifcr,  128;  184;  186. 
of  swine  erysipelas,  112. 
of  swine  plague,  110. 
Bacillus  of  symptomatic  anthrax,  152. 
tetani,  40;  156;  172;  198. 
tuberculosis,  114. 
of  typhoid  fever.  122. 
typhosus,  42;  46;  50;  122:    188;  192; 

200. 
vulgaris,  52:  72. 


Bacteria,    in     air.     80;    transmission 

thiough  the  mail,  200. 
Bacteriological  analysis,  80;  diagnosis, 

178. 
Bacterium  anthracis,  40;  104;  162;  184. 

cuniculicida,  110;  174. 

dipHtheriae.  30;  118;  164;  172;    178; 
200. 

injluenzae,  120;  182;  186. 

leprae,  172;  174;  198. 

mallei,  116;  164;  174;  186;  196. 

phosphorescens,  68. 

pneumoniae,  106;  162;  172:  174;  184; 
186;  196. 

pneumonicum,  108;  162. 

pseudo-diphtheriae,  180. 

rhusiopalhiae,  112;  172. 

tuberculosis.  114;  162;  172;  174:  182; 
186;  192;  196;  200. 

welchii,  150;  196. 
Bismarck  brown,  18. 
Blank  for  animal  experiments,  168. 
Blood,  examination  of,  184. 
Blood  serum,  character  of  growth  on, 

59;    collection    of,    184;    Loeffler's 

mixture,  88. 
Bouillon,  character  of  growth   in,  58; 

glucose,  44;  preparation  of,  4. 
Brownian  movement,  24. 
Buccal  secretions,  examination  of,  178. 
Bunge's  flagella  stain,  40. 

CANADA  balsam,  22. 
Capsule  stain,  42. 

Carbol-fuchsin,  18. 

Cell  grouping,  study  of,  SO. 

Chemicals,  effect  on  bacteria,  48. 

Cholera  red,  192. 

Cholera  vibrio,  132. 

Classification  of  bacteria,  60. 

Cleaning  glassware,  2. 

Colon  bacillus,  16.    See  B.  coli. 

Color  production,  variation  in,  58. 

Coloring  matter,  separation  of,  67. 

Comma  bacillus,  132. 

Concentration  of  media,  effect  on  bac- 
terial growth,  46. 

Cover-glass  preparation,  20. 

Cover-glass,  cleaning  of,  18. 

Culture  characters,  description  of,  57. 

Cultures,  fluid,  16;  incubation  of,  16; 
stab,  16;  streak,  16,  test-tube,  14. 

Culture  media,  care  of,  14;  preparation 
of  4;  10;  12;  44;  63;  88;  steriliza- 
tion, 8. 

DECOLORIZING  agents,  use,  36. 
Desiccation,  effect,  48. 
Diplococcus   of    cerebro-spinal  men- 
ingitis, 100. 
of  gonorrhoea,  98. 
of  pneumonia,  106. 

[203] 


Disinfectant,  48. 
Disinfectants,  testing,  88. 
Drawing  bacteria,  28. 
Dunham's  solution,  44, 
Dust,  relation  of  bacteria  to,  82. 

CBERTH'S  bacillus,  122. 

"    Ehrlich's  anilin  oil  gentian  violet 

18. 

Eisner's  medium,  190. 
Embedding  tissue,  170. 
Endospores,  staining,  38;  study  of,  40 
Enzymes,  54. 
Esmarch  rolls,  34. 

CAECES,  examination  of,  188. 

Fermentation  tube,  50. 
Filter  for  gelatin,  10. 
Flagella  stain,  40. 
Fluid  cultures,  16. 
Form  types,  study  of,  26. 
Fraenkel's  soil  borer,  82. 
Frost's  gasometer,  50. 
Fuchsin,  carbol,  18;  Ziehl's,  18, 

pABBETT'S  methylen  blue.  20;  tu- 

*J    bercle  stain,  S8. 

Gas  analysis,  50;  detection,  50. 

Gasometer,  50. 

Gelatin  glucose,  44;  preparation,  10; 
sterilization,  10. 

Gelatin  plate  cultures,  character  of 
colonies  on  57;  preparation,  32. 

Gelatin  stab  culture,  character  of 
growth  in,  58;  inoculation,  16. 

Gentian  violet,  18. 

Glassware,  cleaning  and  steriliza- 
tion, 2. 

Glucose  media,  44. 

Golden  pus  coccus,  98. 

Gonococcus,  98. 

Gram's,  iodine  solution.  20;  stain,  38. 

H^MATOXYLIN  and  eosin  stain. 
172. 

Hanging-drop  preparation,  24. 
Mauser's  spore  stain,  40. 
Hay  bacillus,  16. 
j     Heat,  effect  on  bacteria,  48. 
[    Hiss'  media,  preparation,  190;  use,  190- 
200. 


IMPRESSION  preparation,  30. 
*     Incubators,  16. 
Indol,  54. 

Involution  forms,  30. 
Iodine  solution,  Gram's  20;  Weigert's 
172. 

JT-LEBS-LOEFFLER  bacillus,    118. 
!»-    Koch's  method  of  air  analysis.  80. 


Index. 


205 


LABELS,  10. 
Lactose  agar,  44. 
Litmus,  lactose  agar-plate,  52;  milk. 

44:  solution,  44;  52. 

Loeffler's  blood  serum,  88;  tissue 
stain,  172. 

MAILING  bacteria, 200. 
Methylen    blue,    Gabbett's,    20; 
Loeffler's,  20. 
Micrococcus  aureus,  96;  172. 

gonorrhotae,  98;  174;  192;  194. 

inlracellularis,  100;  196. 

lanceolatus,  106. 

melilensis,  94. 

pyogmes,  92;  172. 

tetragenus,  102. 

Micrometer,  ocular  and  stage,  28. 
Microscope,  use, 2*2. 
Micros/lira  comma,  132;  190;  200. 

finkleri,  136. 

metschnitovi,  26;  134 
Milk,  character   of   growth   in,  58;  ex- 
amination for  pathogenic  bacteria, 

200;  litmus.  44;  pasteurization,  84; 

quantitative  analysis,  84. 
Monilia  Candida,  180. 
Morphological  characters,  59. 
Movement,  study  of,  24. 

NEISSER'S  diphtheria  stain,  178. 
Nitrites,  detection,  52. 
Nitrate  solution,  44. 
Non-pathogenic  bacteria,  63. 

OBSERVATION  of  inoculated 'ani- 
mals, 164. 

Oil-immersion  objective,  22. 
Oxygen,  effect  on  bacteria,  50. 

nARIETTrS  method,  188;  200. 

*       Pasteurization  of  milk,  84. 

Pathogenic  aerobes,  88;  anaerobes,  149; 
bacteria  in  water  and  food  sup- 
plies, 200. 

Petri  dishes,  32. 

Petri-Sedgwick's  air  analysis,  80. 


Phenolphthalein,  6. 

Physiological  characters,  59. 

Pigment,  production,  56;  varieties,  66. 

Pipettes,  sterilization,  4. 

Plasmodium  malariae,  186. 

Plate  cultures,  gelatin,  32;  agar,  34; 
study,  36. 

Platinum  needles,  14. 

Plugging  flasks  and  tubes,  2. 

Pneumococcus,  106. 

Post-mortem,  examination,  164. 

Potato,  character  of  grown  on,  58;  in- 
oculation, 16;  preparation,  12. 

Proteus  vulgaris,  72. 

Pseudomonas  aeruginosa,  54;  130;  196. 
erythro  sporus,  40. 
fluorescens,  26. 

Pyogenic  micrococci.  180;  186;  192;  194. 

RABIES,  diagnosis,  198. 
Reaction  of  media,  6;   effect  on 
growth  of  bacteria,  44. 
Roll  cultures,  34. 
Russell's  water  sampler,  82. 

SARCINA  lulea,  26. 
tetragena,  102;  162;  172;  174. 

Sections,  cutting,  170;  staining,  172. 

Shake  culture,  50. 

Slides,  cleaning,  18. 

Soap  stone  for  cooling  plate  cultures, 
32. 

Soil,  analysis,  82. 

Spirillum  of  Finkler  and  Prior.  138. 
otermeitri,  186;  192. 
rubrum,  26. 

Sputum,  180. 

Stab  culture,  16, 

Stain  bottles,  20. 

Staining  solutions,  18, 

Staphylococcus  epidermidis  albus,  92. 
pyogenes  albus,  92; aureus,  96. 

Steam  sterilizers,  8. 

Sterilization,  culture  media,  8;  discon- 
tinuous, 8;  gelatin,  10;  glassware, 2; 
instruments.  Irt4. 


Sterilizers,  Arnold,  8;  hot  air,  2;  sim- 
ple form,  8. 

Streak  cultures,  character  of  growth 
on,  58;  inoculation,  16. 

Streptococcus  pyogencs, yd;  162;  194. 

Streptothrix  actinomyces,  172;  184;  198. 

Study  of  bacteria,  57. 

Sugar  media,  preparation,  44;  sterili- 
zation, 8;  44. 

Sulphuretted  hydrogen  in  cultures,  54. 

Sunlight,  effect  on  bacteria,  50. 

TAXONOMY,  56. 
*      Temperature  variations,  effect  on 
bacteria,  46. 

Test-tube  cultures,  inoculation,  14;  26; 
study,  26. 

Test-tubes,  cleaning,  2;  filling,  6. 

Thermal  death  point  determinations, 
46. 

Thermostats,  16. 

Tissue,  embedding,  170;  hardening,  170; 
staining,  172. 

Transudates  and  exudates,  examina- 
tion of,  194. 

Tubercle  stain,  38. 

Typhoid  blood,  Widal  reaction,  188. 

I  TRINE,  examination  of,  192. 


V 


I  BRIO  metschnikovi.  184. 
Vital  movement,  24. 


WATER  analysis.  82;  examination 
for  pathogenic  bacteria,  200. 
Water  blanks,  12. 
Weigert's  stain,  172. 
Welch's  capsule  stain,  42. 
Wertheim's  medium  for  gonococcus, 

99;  194. 

Widal  reaction,  188. 
Wurtz's  lactose  litmus  agar  plate,  52 

ZIEHL'S  carbol  fuchsin,  18. 
Ziehl-Neelsen  stain,  182. 


, 


MAR  20    1901 


143 1 4 


268429 

£T 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


